Coxsackieviruses require phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) for replication but can bypass this need by an H57Y mutation in protein 3A (3A-H57Y). We show that mutant coxsackievirus is not outcompeted by wild-type virus during 10 passages in vitro. In mice, the mutant virus proved as virulent as wild-type virus, even when mice were treated with a PI4KIIIβ inhibitor. Our data suggest that upon emergence, the 3A-H57Y mutant has the fitness to establish a resistant population with a virulence similar to that of wild-type virus.