The non-structural protein-1 (NS1) of influenza A virus binds the p85β subunit of phosphoinositide 3-kinase (PI3K) to induce PI3K activity in the infected cells. Some virus strains encode NS1 containing a motif that binds tightly to the SH3 domain of the cellular adapter proteins Crk and CrkL to potentiate NS1-induced PI3K activation. Here we show that this potentiation involves reorganization of the natural CrkL-p85β complex into a novel trimeric complex where NS1 serves as a bridging factor. Of note, NS1 proteins that lack the SH3 binding capacity can also associate with CrkL, but in a less stable trimeric complex mediated by p85β. The data presented here establish Crk proteins as general host cell cofactors of NS1, and show that the enhanced PI3K activation by SH3 binding-competent NS1 variants is mediated by a more efficient tethering of Crk proteins to the NS1-PI3K complex.