Aim: Recent studies have demonstrated that mesenchymal stem cells (MSCs) can differentiate into endothelial cells. The effect of shear stress on MSC differentiation is incompletely understood, and most studies have been based on two-dimensional systems. We used a model of tissue-engineered vascular grafts (TEVGs) to investigate the effects of shear stress on MSC differentiation. Methods: MSCs were isolated from canine bone marrow. The TEVG was constructed by seeding MSCs onto poly- caprolactone and lactic acid (PCLA) scaffolds and subjecting them to shear stress provided by a pulsatile bioreactor for four days (two days at 1 dyne/cm 2 to 15 dyne/cm 2 and two days at 15 dyne/cm 2). Results: Shear stress significantly increased the expression of endothelial cell markers, such as platelet-endothelial cell adhesion molecule-1 (PECAM-1), VE-cadherin, and CD34, at both the mRNA and protein levels as compared with static control cells. Protein levels of alpha-smooth muscle actin (α-SMA) and calponin were substantially reduced in shear stress-cultured cells. There was no significant change in the expression of α-SMA, smooth muscle myosin heavy chain (SMMHC) or calponin at the mRNA level. Conclusion: Shear stress upregulated the expression of endothelial cell-related markers and downregulated smooth muscle-related markers in canine MSCs. This study may serve as a basis for further investigation of the effects of shear stress on MSC differentiation in TEVGs. © 2009 CPS and SIMM.