The laminin family of proteins is critical for managing a variety of cellular activities including migration, adhesion, and differentiation. In bone, the roles of laminins in controlling osteogenic differentiation of human mesenchymal stem cells (hMSC) are unknown. We report here that laminin-5 is found in bone and expressed by hMSC. hMSC isolated from bone synthesize laminin-5 and adhere to exogenous laminin-5 through α3β1 integrin. Adhesion to laminin-5 activates extracellular signal-related kinase (ERK) within 30 min and leads to phosphorylation of the osteogenic transcription factor Runx2/CBFA-1 within 8 d. Cells plated on laminin-5 for 16 d express increased levels of osteogenic marker genes, and those plated for 21 d deposit a mineralized matrix, indicative of osteogenic differentiation. Addition of the ERK inhibitor PD98059 mitigates these effects. We conclude that contact with laminin-5 is sufficient to activate ERK and to stimulate osteogenic differentiation in hMSC.