Matrix elasticity directs stem cell lineage specification. Mesenchymal stem cells (MSCs) respond to matrix stiffness, with soft matrices mimicking brain promoting neurogenesis, stiffer matrices mimicking muscle promoting myogenesis, and rigid matrices mimicking bone promoting osteogenesis. Lineage commitment is influenced by matrix elasticity, with cells committing to a lineage after several weeks in culture. Inhibition of nonmuscle myosin II blocks elasticity-directed lineage specification without significantly affecting other cell functions. The study shows that matrix stiffness is a key factor in stem cell differentiation, with lineage specification dependent on matrix elasticity and nonmuscle myosin II activity. MSCs on soft matrices express neurogenic markers, while those on stiffer matrices express myogenic or osteogenic markers. Induction factors can reprogram lineages initially, but long-term commitment is determined by matrix elasticity. The results highlight the importance of matrix stiffness in stem cell differentiation and have implications for understanding the in vivo microenvironment and stem cell therapies.Matrix elasticity directs stem cell lineage specification. Mesenchymal stem cells (MSCs) respond to matrix stiffness, with soft matrices mimicking brain promoting neurogenesis, stiffer matrices mimicking muscle promoting myogenesis, and rigid matrices mimicking bone promoting osteogenesis. Lineage commitment is influenced by matrix elasticity, with cells committing to a lineage after several weeks in culture. Inhibition of nonmuscle myosin II blocks elasticity-directed lineage specification without significantly affecting other cell functions. The study shows that matrix stiffness is a key factor in stem cell differentiation, with lineage specification dependent on matrix elasticity and nonmuscle myosin II activity. MSCs on soft matrices express neurogenic markers, while those on stiffer matrices express myogenic or osteogenic markers. Induction factors can reprogram lineages initially, but long-term commitment is determined by matrix elasticity. The results highlight the importance of matrix stiffness in stem cell differentiation and have implications for understanding the in vivo microenvironment and stem cell therapies.