If you had 5 nights in Tuscany.........

Closer to Assisi, we loved Cortona, Spello, and Spoleto. We also loved the Crete Senses region with Montepulciano, San Quirico d'Orcia, and Pienza as our favs.

Those have been our favorites outside of the Chianti/Lucca/Orvieto area.

After visiting a dozen villages one year, we met someone who told us their favorite town was Todi - and we had not even driven there !

We have found that a lovely meal, a serendipitous meeting with an Italian family, and just a delicious cappuccino has swayed our delight in any given village !

Montepulciano-Pienza-Montalcino is probably on top for me, also because of the car ride. This part of Tuscany is just wonderful.
When in Montalcino, you can also visit the Sant'Antimo Abbey not too far.
Volterra is another great one.
Very small but very pretty, Monterigionni.
I find San Gimignano too touristy but it's still a beautiful small town. Go esrly morning or at night. Less crowded.
Cortona was charming.
Gubbio is not too far from Assisi and you have the feeling of walking in streets that our 700 years old.

Montalcino and Arezzo. Several places listed above are in Umbria, which I prefer to Tuscany.

North-West of Siena (in order of itinerary sequence):
Monteriggioni-Colle Val D'Elsa-San Gimignano-Volterra

South of Siena:
Buonconvento, Montalcino, San Quirico, Pienza, Monticchiello, Montepulciano

With only 2 nights in Assisi you may not have time for a lot more.
On the way there, maybe you can stop in Cortona. Spello is very close to Assisi, so you might squeeze it in. Gubbio is at least 45 min from Assisi and it deserves at least half day. If you can fit it in your schedule, go for it. It's great.

We stayed in Arezzo for 7 nights and there were many cute towns to visit from there. A favorite was Montalcino.
We liked Orievto but it is Umbria. I thought Cortona was a wasted day

Karen,
This may help http://www.slowtrav.com/italy/tuscany/hs_planning.htm