is Adjustment Location required when uploading?

It is. And you can only do one location at a time (in my experience).

the Adjustment location field at the header level?

Header and line.

In our instance, the Adjustment Location in the header is not mandatory because this is used just to tag the Adjustment Account and not for inventory (line level). You may want to check your form, script or workflow or simply view it in the UI if it's mandatory or not.

I would suggest both levels so it shows in financial reports properly