I've been playing around with a similar setup. When installing Linux on the flash drive, I unplugged the hard drive from the mobo before installing from the Live CD to prevent any accidental formatting or screwing up of the main hard drive.

Also, when running from USB I believe my hard drive did mount automatically, so if you're just playing around with Linux and don't plan to access any files on the hard drive, I would take the extra step of keeping the hard drive unmounted. Don't know how but I'm sure there is a setting somewhere to not mount upon boot-up.