What are the differences ?
Why needed GUI 5.0 ?

There is no difference in the output file except for the header to define the version.

Where it matters is for Windows to load the file. If you say 5.0 but your Windows only supports 4.0 then it won't load or run.

Windows may enable some extra features, or link the code with different system DLL versions.

If thour program targeted Vista+ and use more extended structures for common controls than in official package thou could use PE GUI 6.0 so program will not crash but notice user that it for other OS.
If thou use unicode headers so that will be not honesty to use PE GUI 4.0 because there is no support for unicode in win95 (4.0 is win95)
if thour app is 64 bit GUI 5.0 is minimum.

For every windows versions size of some common controls are different - there is needed research to prove fact that according to requested GUI version Windows will load appropriate COMCTL.DLL.

I suspect if thou use structures from fasm package and define GUI 6.0 program will crash on use of some common controls.

NT 4.0 is also, well, 4.0.