The study investigates the role of substrate stiffness in the differentiation of myoblasts into myotubes, a process crucial for muscle function. Myoblasts were cultured on collagen-coated glass or polymer gels of varying stiffness, and their subsequent fusion into myotubes was observed. The results show that while myotube formation is independent of substrate flexibility, the development of myosin/actin striations, a key indicator of muscle differentiation, is significantly influenced by the stiffness of the substrate. Myotubes formed on gels with stiffness similar to normal muscle tissue (passive Young’s modulus of about 12 kPa) showed robust striations, whereas those on softer or stiffer gels, including those mimicking stiff dystrophic muscle, did not. Additionally, myotubes grown on top of compliant glass-attached myotubes (but not on softer fibroblasts) exhibited striations, while the bottom cells only assembled stress fibers and vinculin-rich adhesions. The study also found that adhesion strength increases monotonically with substrate stiffness, with the strongest adhesion observed on glass. These findings have significant implications for the introduction of stem cells into diseased or damaged striated muscle, emphasizing the importance of substrate stiffness in promoting optimal differentiation.The study investigates the role of substrate stiffness in the differentiation of myoblasts into myotubes, a process crucial for muscle function. Myoblasts were cultured on collagen-coated glass or polymer gels of varying stiffness, and their subsequent fusion into myotubes was observed. The results show that while myotube formation is independent of substrate flexibility, the development of myosin/actin striations, a key indicator of muscle differentiation, is significantly influenced by the stiffness of the substrate. Myotubes formed on gels with stiffness similar to normal muscle tissue (passive Young’s modulus of about 12 kPa) showed robust striations, whereas those on softer or stiffer gels, including those mimicking stiff dystrophic muscle, did not. Additionally, myotubes grown on top of compliant glass-attached myotubes (but not on softer fibroblasts) exhibited striations, while the bottom cells only assembled stress fibers and vinculin-rich adhesions. The study also found that adhesion strength increases monotonically with substrate stiffness, with the strongest adhesion observed on glass. These findings have significant implications for the introduction of stem cells into diseased or damaged striated muscle, emphasizing the importance of substrate stiffness in promoting optimal differentiation.