Hepatocyte growth factor (HGF) and its receptor, Met, play a crucial role in regulating adult skeletal myoblast proliferation and differentiation. Met signaling is mediated by phosphorylation of two carboxy-terminal tyrosines, which act as docking sites for a number of intracellular mediators. These include Grb2 and p85, which couple the receptor with the Ras and phosphatidylinositol 3-kinase (PI3K) pathways, respectively. In this study, we define the role of these effectors in response to HGF by utilizing Met mutants, designed to obtain preferential coupling of Met to either Grb2 or PI3K or both. We found that relative to the wild-type receptor, enhanced binding to Grb2 further increases the incorporation of bromodeoxyuridine and the expression of Twist, while decreasing that of p27Kip1 and myogenin. Conversely, preferential coupling with PI3K induced cell-cycle withdrawal and differentiation. Whereas enhanced Grb2 binding increased the phosphorylation of the mitogen-activated protein kinase/extracellular signal-regulated protein kinases (MAPK/ERK) and abrogated that of p38 MAPK, PI3K had the opposite effect. PD098059 reversed the inhibitory effects of Met on cell proliferation and differentiation, while wortmannin had only a very marginal effect. Taken together, these data suggest that coupling of Met with Grb2 is necessary for HGF-mediated inhibition of muscle differentiation. This inhibition occurs only when PI3K signaling downstream of Met is low. Imposing an efficient coupling of PI3K to Met would lead to up-regulation of muscle regulatory factors and subsequent cell differentiation.