PSTPIP is a tyrosine phosphorylated protein involved in the organization of the cytoskeleton. Its ectopic expression induces fillipodial-like membrane extensions in NIH 3T3 cells. We previously observed a defect in cytokinesis and an increase in the tyrosine phosphorylation of the adapter protein PSTPIP in PTP-PEST deficient fibroblasts. In this manuscript, we demonstrate that PTP-PEST and PSTPIP are found in the same complexes in vivo and that they interact directly through the CTH domain of PTP-PEST and the coiled-coil domain of PSTPIP. We tested several pathways that could regulate the tyrosine phosphorylation of PSTPIP. We found that the activation of the EGF and PDGF receptors can induce PSTPIP phosphorylation. With the use of the PP2 inhibitor, we demonstrate that Src-kinases are not involved in the EGF-mediated phosphorylation of PSTPIP. Together with previous results, this suggests that c-Abl is the critical tyrosine kinase downstream of growth factor receptors responsible for PSTPIP phosphorylation. We also demonstrate that PTP-PEST dephosphorylates PSTPIP at tyrosine 344 and this event regulates the association between SH2 domain-containing proteins and PSTPIP. Importantly, we identified tyrosine 344 as the main phosphorylation site of PSTPIP by performing tryptic phosphopeptide maps. This is an important finding since tyrosine 367 of PSTPIP was also proposed as a candidate phosphorylation site involved in the negative regulation of the association between PSTPIP and WASP. In this respect, we observed that the PSTPIP-WASP complex is stable in vivo and is not affected by the phosphorylation of PSTPIP. Furthermore, we demonstrate that PSTPIP serves as a scaffold protein between PTP-PEST and WASP and allows PTP-PEST to dephosphorylate WASP. This finding suggest a possible mechanism for PTP-PEST to directly modulate actin remodling through the PSTPIP-WASP interaction.