Myotubes differentiate optimally on substrates with tissue-like stiffness, which has significant implications for muscle diseases and tissue engineering. This study shows that myoblasts cultured on collagen-coated substrates of varying stiffness form myotubes, but only those with stiffness similar to normal muscle (around 12 kPa) develop striations, which are essential for muscle function. On softer or stiffer substrates, myotubes do not develop striations, indicating that substrate stiffness is crucial for proper muscle differentiation. The study also found that myotubes grown on a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) develop striations, while the bottom cells only form stress fibers and adhesions. This suggests that substrate stiffness influences not only the formation of myotubes but also their ability to develop contractile structures. The research highlights the importance of substrate stiffness in muscle differentiation, as it affects cell adhesion, spreading, and the formation of myofibrils. The optimal stiffness for myotube differentiation is similar to that of normal muscle, and deviations from this stiffness hinder the development of striations. This finding has important implications for tissue engineering and regenerative medicine, as it suggests that the mechanical properties of the substrate can significantly influence the differentiation of stem cells into functional muscle cells. The study also demonstrates that the mechanical properties of the extracellular matrix (ECM) can influence the differentiation of muscle cells. For example, in dystrophic muscle, the ECM is stiffer, which may hinder the differentiation of stem cells into functional muscle cells. Similarly, in atherosclerosis, the ECM is altered, which may affect the function of smooth muscle cells. The results of this study suggest that the mechanical properties of the substrate are a critical factor in the differentiation of muscle cells. This has important implications for the development of tissue-engineered muscle and for understanding the pathogenesis of muscle diseases. The findings also highlight the importance of substrate stiffness in the differentiation of other cell types, such as fibroblasts and stem cells, and suggest that the mechanical properties of the substrate can be used to control cell behavior in tissue engineering applications.Myotubes differentiate optimally on substrates with tissue-like stiffness, which has significant implications for muscle diseases and tissue engineering. This study shows that myoblasts cultured on collagen-coated substrates of varying stiffness form myotubes, but only those with stiffness similar to normal muscle (around 12 kPa) develop striations, which are essential for muscle function. On softer or stiffer substrates, myotubes do not develop striations, indicating that substrate stiffness is crucial for proper muscle differentiation. The study also found that myotubes grown on a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) develop striations, while the bottom cells only form stress fibers and adhesions. This suggests that substrate stiffness influences not only the formation of myotubes but also their ability to develop contractile structures. The research highlights the importance of substrate stiffness in muscle differentiation, as it affects cell adhesion, spreading, and the formation of myofibrils. The optimal stiffness for myotube differentiation is similar to that of normal muscle, and deviations from this stiffness hinder the development of striations. This finding has important implications for tissue engineering and regenerative medicine, as it suggests that the mechanical properties of the substrate can significantly influence the differentiation of stem cells into functional muscle cells. The study also demonstrates that the mechanical properties of the extracellular matrix (ECM) can influence the differentiation of muscle cells. For example, in dystrophic muscle, the ECM is stiffer, which may hinder the differentiation of stem cells into functional muscle cells. Similarly, in atherosclerosis, the ECM is altered, which may affect the function of smooth muscle cells. The results of this study suggest that the mechanical properties of the substrate are a critical factor in the differentiation of muscle cells. This has important implications for the development of tissue-engineered muscle and for understanding the pathogenesis of muscle diseases. The findings also highlight the importance of substrate stiffness in the differentiation of other cell types, such as fibroblasts and stem cells, and suggest that the mechanical properties of the substrate can be used to control cell behavior in tissue engineering applications.