Extracellular matrix (ECM) stiffness regulates the lineage commitment of mesenchymal stem cells (MSCs). Although cells sense ECM stiffness through focal adhesions, how cells sense ECM stiffness and regulate ECM stiffness-dependent differentiation remains largely unclear. In this study, we show that the cytoskeletal focal adhesion protein vinculin plays a critical role in the ECM stiffness-dependent adipocyte differentiation of MSCs. ST2 mouse MSCs differentiate into adipocytes and osteoblasts in an ECM stiffness-dependent manner. We find that a rigid ECM increases the amount of cytoskeleton-associated vinculin and promotes the nuclear localization and activity of the transcriptional coactivator paralogs Yes-associated protein (YAP, also known as YAP1) and transcriptional coactivator with a PDZ-binding motif (TAZ, also known as WWTR1) (hereafter YAP/TAZ). Vinculin is necessary for enhanced nuclear localization and activity of YAP/TAZ on the rigid ECM but it does not affect the phosphorylation of the YAP/TAZ kinase LATS1. Furthermore, vinculin depletion promotes differentiation into adipocytes on rigid ECM, while it inhibits differentiation into osteoblasts. Finally, TAZ knockdown was less effective at promoting adipocyte differentiation in vinculin-depleted cells than in control cells. These results suggest that vinculin promotes the nuclear localization of transcription factor TAZ to inhibit the adipocyte differentiation on rigid ECM.