You shouldn't have any trouble ... but before doing the upgrade I recommend you do the following:
- Back up your theme
- Back up all of your plug-ins
- Back up your existing database
This will allow you to "undo" the upgrade if needed.
Check theme compatibility
If you're using a commonly available theme, check to make sure it's compatible with WordPress 3.0.1 before upgrading. Chances are good that it will be, but if you can double check before upgrading you can save yourself a potential headache. I've seen people try to upgrade from version 2.0 to version 2.9 with themes that broke afterwards ...
Check plug-in compatibility
Some plug-ins might not work with the new version of WordPress. Check to see if they are rated as compatible before upgrading - if they're not, be prepared for a plug-in to potentially break ... so if it's an absolute necessity, look for a potential alternative in the event that it doesn't work.
I've had various calendar plug-ins fail on new version of WordPress ... so make sure you have an option available if something goes wrong.
Be prepared for everything to work anyway
I've upgraded several systems from 2.7.X to 3.0 over the past few weeks without any problems. So there's a very good chance that your update will go off without a hitch and these precautionary steps were a waste of time. But better to waste the time now than to have to fix a broken site after the upgrade.
Upgrade your plug-ins one-at-a-time. If you're upgrading a whole slew of things at once, it can be harder to track down what caused any errors (if any do occur). Upgrading plug-ins one-at-a-time will help you isolate and fix any problems. If a plug-in doesn't work after the upgrade, you can always go back to the back-up you made earlier.