The study investigates the role of matrix elasticity in directing the lineage specification of naive mesenchymal stem cells (MSCs). MSCs exhibit extreme sensitivity to tissue-level elasticity, with softer matrices mimicking brain environments promoting neurogenic differentiation, stiffer matrices mimicking muscle environments leading to myogenic differentiation, and even stiffer matrices mimicking collagenous bone environments fostering osteogenic differentiation. During the initial week in culture, soluble induction factors can reprogram these lineages, but after several weeks, the cells commit to the lineage specified by matrix elasticity. Inhibition of nonmuscle myosin II blocks elasticity-directed lineage specification without significantly affecting other aspects of cell function and shape. The findings highlight the importance of physical effects of the in vivo microenvironment and have implications for therapeutic uses of stem cells.The study investigates the role of matrix elasticity in directing the lineage specification of naive mesenchymal stem cells (MSCs). MSCs exhibit extreme sensitivity to tissue-level elasticity, with softer matrices mimicking brain environments promoting neurogenic differentiation, stiffer matrices mimicking muscle environments leading to myogenic differentiation, and even stiffer matrices mimicking collagenous bone environments fostering osteogenic differentiation. During the initial week in culture, soluble induction factors can reprogram these lineages, but after several weeks, the cells commit to the lineage specified by matrix elasticity. Inhibition of nonmuscle myosin II blocks elasticity-directed lineage specification without significantly affecting other aspects of cell function and shape. The findings highlight the importance of physical effects of the in vivo microenvironment and have implications for therapeutic uses of stem cells.