Are there regulations regarding customers in the back room of a 501c3 thrift store?

Doing what exactly? Having poker games? Delivering donations?

Having poker games?

Love it!

I suspect the concern is customers having access to items that have been recently donated and are not yet for sale to the general public. If that's the issue, then no, there are no regulations. It would be similar to having a private sale. The only concern would be if the items are sold from the back room at lower price than the 501c3 could otherwise get if the items were sold in the store front - if this is the case store management and the 501c3 board should set clear rules that govern this practice, as it is costing the 501c3 money. And if items are sold on consignment (i.e. where the donor gets a portion of the proceeds) this may be a touchy subject.

Of if the store, allowed some customers to pick out items before they become publicly available.

But a store has to have management rules. For safety and security, customers should only be in the public area.