ONYX-015 is an adenovirus that lacks the E1B-55K gene product for p53 degradation. Thus, ONYX-015 was conceived as an oncolytic virus that would selectively replicate in p53-defective tumor cells. Here we show that loss of E1B-55K leads to the induction, but not the activation, of p53 in ONYX-015-infected primary cells. We use a novel adenovirus mutant, ONYX-053, to demonstrate that loss of E1B-55K-mediated late viral RNA export, rather than p53 degradation, restricts ONYX-015 replication in primary cells. In contrast, we show that tumor cells that support ONYX-015 replication provide the RNA export function of E1B-55K. These data reveal that tumor cells have altered mechanisms for RNA export and resolve the controversial role of p53 in governing ONYX-015 oncolytic selectivity.