Activation of PKCalpha will induce the cSrc binding partner, AFAP-110, to colocalize with and activate cSrc. The ability of AFAP-110 to colocalize with cSrc is contingent upon the integrity of the amino terminal Pleckstrin Homology (PH1) domain, while the ability to activate cSrc is dependent upon the integrity of its SH3 binding motif, which engages the cSrc SH3 domain. The outcome of AFAP-110-directed cSrc activation is a change in actin filament integrity and the formation of podosomes. Here, we address what cellular signals promote AFAP-110 to colocalize with and activate cSrc, in response to PKCalpha activation or PMA treatment. As PH domain integrity in AFAP-110 is required for colocalization and PH domains are known to interact with both protein and lipid binding partners, we sought to determine whether PI3K activation played a role in PMA induced colocalization between AFAP-110 and cSrc. We show that PMA treatment is able to direct activation of PI3K. Treatment of mouse embryo fibroblast with PI3K inhibitors blocked PMA-directed colocalization between AFAP-110 and cSrc and subsequent cSrc activation. PMA was also unable to induce colocalization or cSrc activation in cells that lacked the p85alpha and beta regulatory subunits of PI3K. This signaling pathway was required for migration in a wound healing assay. Cells that were null for cSrc or the p85 regulatory subunits or expressed a dominant-negative AFAP-110 also displayed a reduction in migration. Thus, PI3K activity is required for PMA-induced colocalization between AFAP-110 and cSrc, subsequent cSrc activation and this signaling pathway promotes cell migration.