Human mesenchymal stem cells (hMSCs) are characterized by their abilities to differentiate into different lineages, including osteoblasts. Besides soluble factors, mechanical strain and extracellular matrix (ECM) proteins play important roles in osteogenic differentiation of hMSCs. However, interactions between them are still not fully understood. The purpose of this study was to investigate the combined effects of insoluble chemical and mechanical factors (ECM proteins vs. cyclic stretching) in driving hMSCs into osteogenic differentiation. To avoid the influence from osteogenic supplements, hMSCs were cultured in regular medium and subjected to cyclic mechanical stretching using a Flexcell Tension system (3% elongation at 0.1 Hz) when they were grown on substrates coated with various ECM proteins (collagen I (Col I), vitronectin (VN), fibronectin (FN), and laminin (LN)). Using alkaline phosphatase (ALP) activity and mineralized matrix deposition as respective indicators of the early and late stages of osteogenesis, we report herein that all of the ECM proteins tested supported hMSC differentiation into osteogenic phenotypes in the absence of osteogenic supplements. Moreover, cyclic mechanical stretching activated the phosphorylation of focal adhesion kinase (FAK), upregulated the transcription and phosphorylation of core-binding factor alpha-1 (Cbfa1), and subsequently increased ALP activity and mineralized matrix deposition. Among the ECM proteins tested, FN and LN exhibited greater effects of supporting stretching-induced osteogenic differentiation than did Col I and VN. The ability of ECM proteins and mechanical stretching to regulate osteogenesis in hMSCs can be exploited in bone tissue engineering via approximate matrix design or application of mechanical stimulation. J. Cell. Biochem. 108: 1263–1273, 2009. © 2009 Wiley-Liss, Inc.