Throughout gestation, fetal growth and development depend, in part, on placental transfer of nutrients from the maternal circulation. This latter function depends on multinucleated, terminally differentiated syncytiotrophoblasts. In vitro, freshly isolated cytotrophoblast cells differentiate spontaneously into syncytiotrophoblast in the presence of fetal bovine serum (FBS). We have previously showed that trophoblast differentiation is regulated by ERK1/2 and p38. Moreover, we showed that PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d]pyrimidine], a Src family kinase (SFK) specific inhibitor, stimulates biochemical trophoblast cells differentiation while it inhibits cell adhesion and spreading without affecting cell fusion. Therefore, we examined the mechanisms by which PP2 modulates trophoblast cells differentiation. This study shows that PP2 stimulates ERK1/2 and p38 activation after 24h of treatments and up to 3 days while it inhibits focal adhesion kinase (FAK) phosphorylation at many sites including Tyr-397, 407, 576 and 577. Furthermore, we showed that transient activation of ERK1/2 by FBS is independent of SFK and that PP2 induces rapid activation of p38. Moreover, the kinase activity of SFK is negatively regulated by the phosphorylation of their carboxy (C)-terminal regulatory tyrosines by specific proteins called carboxyl-terminal Src kinase (Csk) and Csk homologous kinase (CHK). We showed the expression of Csk and CHK in human trophoblast cells. In summary, this study showed that PP2 stimulates the biochemical differentiation of trophoblast cells by stimulating p38 and ERK1/2 while it inhibits the morphological differentiation by inhibiting FAK activation.