We've seen mostly lists of what to see for your first time, or lists targetted at those going the first time. I'm being selfish as I'm going to these three in August, but thought I'd start a thread of what to make sure you see or do every single time you go to one of these cities. I'll start it off...

Rome
Vatican Museums
Vatican necropolis / scavi tour
Trevi Fountain
Piazza Novona
Piazza del Popola (I love the twin churches)
Pantheon
Spanish Steps (just to see them)
Mass at the Vatican (I'm Catholic)
Colosseum
St. Peter Enchained (for the Moses statue)
Forums
Walk up the Victor Emmanuel (for a great view of the city)
Papal audience (see above)
Domus Aurea
Ostia Antica

Florence
Accademia Museum
Duomo

Venice
Piazza San Marco
Piazza San Marco, at night (much better)
Water taxi up and down the Grand Canal

I would add in Florence:
-Pitti Palace
- Ufizzi Gallery
- Bargello Museum
- Ponte Vechio

You can visit in an afternoon, from Florence, Pisa.

From Venice you can go to Murano.

From Rome you can go in a evening to see Tivoli Gardens.
You can also visit Villa Borgese, try to see as many of Bernini's fountains.
Visit Capitol Museum.

What a beautiful trip you shall have! You will enjoy for sure!

In Venice, I'd add the Accademia and Guggenheim museums, Scuola Grande di San Rocco, Scuola di San Giorgio degli Schiavoni, Ca' d'Oro, Ca' Rezzonico, Museo Correr, San Giorgio Maggiore, and San Zaccaria.

There's a lot of amazing art in Venice's churches.

When P. San Marco is listed, I'm going to assume that includes the Doges' Palace, Campanile, and the Basilica.

Actually for Venice, I don't have a lot planned. The Basillica for sure; the Doge's Palace was something we saw on our first trip and I don't think we'll be going again. That seemed like a one time is enough kind of place for me.

In Florence, The Baptistry.

For me, a second trip to these cities would mean a lot of just plain walking around and cafe sitting!

A visit to Rome for me would not be complete without going to Campo dei Fiori and having a slice of pizza, going to see the cats at Largo Argentina, seeing the Pantheon first thing in the morning, with the light flooding through the center of the dome, and wandering into Santa Maria sopra Minerva and seeing the frescoes by Lippi. Take the bus up to Santa Maria della Vittoria to see the Ecstasy of St. Theresa-it is amazing. Have lunch at Trattoria Monti -they are so friendly there, and lunch is relaxed and filled with locals. Have lots and lots of cappucino in the morning at Tazza d'Oro, near the Pantheon; make sure you have enough room in your suitcase to bring back a bag of their beans. Go to Obika, a mozzarella bar that will blow your mind. I would not go to Rome without having dinner at one of my favorite restaurants, Myosotis. They have a great wine list. And even though it is a bit out of the way, I take a taxi to Checchino dal 1887 for the best meal in Rome. Visit Castel Sant'Angelo, and at dusk, walk across the Ponte Sant'Angelo and experience Bernini's ten sculptures of angels as they are lit up.

In Florence, I would have to insist upon wandering the streets of Oltrarno on the way to Santa Maria della Carmine, then seeing the frescoes in the Brancacci Chapel, which depict the life of St. Peter, then going to Santo Spirito. I have to stop by the great leather shop Roberta and get some gloves or a bag, then I love to have lunch at Trattoria Mamma Gina. It is a must to have a glass of wine at Enoteca Fuori Porta when on that side of the Arno as well. Near San Lorenzo, I love going to the Medici Chapels (Cappelle Medicee) and seeing the Michelango sculptures of Night, Day, Dawn and Dusk -it is always quiet and cool, not on the top of the list of many tourists, and you get to gaze at some of Michelangelo's best sculpture works up close. In that neighborhood, I grab a simple, but very good dinner at Palle D'Oro on Via Sant'Antonino. In the summer (or anytime), have gelato at Perche No and in the autumn or winter, hot chocolate at Vestri. Go to Mercato Sant Ambrogio, and try to hit Forte di Belvedere.

In Venice, you must take the vaporetto to Santa della Salute in the early morning. When you round the corner on the Grand Canal and see it looming before you, it is simply breathtaking. Get lost wandering the streets of Dorsorduro. Have lunch at La Zucca, a cozy, friendly restaurant that serves great food, not typically Venetian. Also great for morning walks is the route from the train station through Canareggio - you will see people going about their daily routine and pass through a great produce market. Speaking of markets, I have to visit the fish market off the Ponte Vecchio - it always helps to see what looks the best that day so you know what to order for dinner at night! Have linquine with clams and a carafe of local wine at Ai 4 Feri in Dorsorduro, and for a fancy meal, don't miss Fiaschetteria Toscana, its not Tuscan food at all, but the freshest fish of day will be featured, and the service is excellent. I always have to find a lace shop that has real Venetian, hand-sewn lace. I'll take that over Murano glass any day. In the early evening, take any of the Vaporetto routes from the train station that go to the Fondamente Nuove or Ospedale stops...then challenge yourself to find your way back. Through those tiny side streets, you will find yourself in little neighborhood campos with children playing, couples strolling, and Venetians sitting on benches, enjoying the late afternoon sun.

faredolce, I want to follow you around!

For me, muuch (tho' not all) of what is mentioned above, plus

Venice Correr Museum

Florence Convent/Museum San Marco

by the way, these are all goals for me, but unless I have two weeks in each place (which I don't) I couldn't really get to all these places on every trip unless I were running around in a frenzy,it's more of a wish list.
As in, I wish I had more time.
Sigh.