I am not familiar with all the issues of Squeezbox-audio or streaming audio in general.
However, for USB and firewire there is connectivity, drivers, CPU activity and USB polling to consider. That is you are probably not getting 400 from firewire 400, and definitely not 480 from USB 2.0 Theoretically firewire offloads the CPU activity to the firewire card, but I am sure drivers could get scewed up. I am not a fan of USB because of the choices they made when designing the protocols - probably mostly academic these days, in the old days you couldn't even burn a CD half the time over USB.
1. DAS (direct access storage) which you seem to be doing, will greatly outperform NAS (network attached storage). Audio and Video streaming should be done from DAS. Was the firewire 800 drive direct attached to your PC, and was the previous drive in the same configuration. (It seems clear but want to clarify)
2. It may be a case of better cable, card interface connectivity, or a better PCI bus interface (HW and or SW) with the firewire 800 setup.
3. As an aside, wireless A/V is probably not ready for prime time and would require a really good wireless router with matching wireless cards, and definitely not the Linksys WRT54G
v5 http://www.smallnetbuilder.com/content/view/26843/51/ (I personnaly just got the D-Link DIR-655, relatively expensive like the new draftN routers but got it for $109 at circuit city - I think today is the last day before it goes back to $149! But again I am not going to do wireless A?V at this point))