This study examined whether focal adhesion kinase (FAK) plays a role in the differentiation of C2C12 myoblasts into myotubes. Differentiation of C2C12 myoblasts induced by switch to differentiation culture medium was accompanied by a transient reduction of FAK phosphorylation at Tyr-397 (to ~50%, at 1 and 2 h), followed by an increase thereafter (to 240% up to 5 days), although FAK protein expression remained unchanged. FAK and phosphorylated FAK were found at the edge of lamellipodia in proliferating cells, while the later increase in FAK phosphorylation in differentiating cells was accompanied by its preferential location at the tip of well organized actin stress fibers. Hyperexpression of FAK autophosphorylation site (Tyr-397) mutant (MT-FAK) reduced FAK phosphorylation at Tyr-397 in proliferating cells and was accompanied by reduction of cyclin D1 and increase of myogenin expression. These cells failed to progress to myotubes in differentiation medium. In contrast, hyperexpression of a wild type FAK construction (WT-FAK) increased baseline and abolished the transient reduction of FAK phosphorylation at Tyr-397 in serum starved C2C12 cells. Cells transfected with WT-FAK failed to reduce cyclin D1 and to increase myogenin expression, as well as to progress to terminal differentiation in differentiation medium. These data indicate that FAK signaling plays a critical role in the control of cell cycle as well as in the progression of C2C12 cells to terminal differentiation. Transient inhibition of FAK phosphorylation at tyrosine 397 contributes to trigger the myogenic genetic program but its later activation is also central to terminal differentiation into myotubes.