In addition to their chemical properties and topographical features, the mechanical properties of gels have been recently demonstrated to play an important role in various cellular processes, which include cell attachment, proliferation, and differentiation. In this work, multilayer films made of poly(L-lysine)/hyaluronan (PLL/HA) of controlled stiffness have been used to investigate the effects of the mechanical properties of thin films on skeletal muscle cell (C2C12 cells) differentiation. Prior to differentiation, the cells need adhere and proliferate in a growth medium. Stiff films (E0 > 320 kPa) promote the formation of focal adhesions and organization of the cytoskeleton as well as an enhanced proliferation, whereas soft films are not favorable for cell anchoring, spreading, or proliferation. C2C12 cells were then switched to a low serum-containing medium to induce cell differentiation, which was also greatly dependent on film stiffness. Although myogenin and troponin T expressions are only moderately affected by the film stiffness, the morphology of the myotubes exhibit striking stiffness-dependent differences. Soft films allow differentiation only for a few days and the myotubes are very short and thick. Cell clumping followed by aggregate detachment is observed after ∼2 to 4 d. On stiffer films, significantly more elongated and thinner myotubes are observed for up to ∼2 weeks. Myotube striation is also observed but only for the stiffer films. These results demonstrate that film stiffness strongly modulates adhesion, proliferation and differentiation, each of these processes having its own stiffness requirement.