AbstractRASSF4, a member of the classical RASSF family of scaffold proteins, is associated with alveolar rhabdomyosarcoma, an aggressive pediatric cancer of muscle histogenesis. However, the role of RASSF4 in normal myogenesis is unknown. We demonstrate here that RASSF4 is necessary for early in vitro myogenesis. Using primary human myoblasts, we show that RASSF4 expression is dramatically increased during in vitro myogenic differentiation, and conversely that RASSF4‐deficient myoblasts cannot differentiate, potentially because of a lack of upregulation of myogenin. In microscopy studies, we show that RASSF4 protein co‐localizes with proteins of the myogenic microtubule‐organizing center (MTOC) both before and after myogenic differentiation. RASSF4‐deficient cells subject to differentiation conditions demonstrate a lack of shape change, suggesting that RASSF4 plays a role in promoting microtubule reorganization and myoblast elongation. In biochemical studies of myotubes, RASSF4 associates with MST1, suggesting that RASSF4 signals to MST1 in the myogenic differentiation process. Expression of MST1 in myoblasts partially reversed the effect of RASSF4 knockdown on differentiation, suggesting that RASSF4 and MST1 coordinately support myogenic differentiation. These data show that RASSF4 is critical for the early steps of myogenic differentiation.