For what it is worth, I prepared a prebuilt AmigaOS 3.1.4 for one of our testers that had an A4000D with a Picasso IV.
I did nothing special besides what I would consider a normal OS deployment, and the tester only had to configure the card screen modes, but everything else worked out of the box.
Maybe in your case it is some third party program that is doing some funky stuff and getting in the way.
PS: The OS when it installs does not create a user-startup file. That one is generated by the user or applications.
