Mesenchymal stem cells (MSCs) have tremendous potential as a cell source for regenerative medicine due to their capacity for differentiation into endothelial-like cells when seeded on nonmodified cover glasses. This absence of removable surface, preventing recovery of cell sheet, constitutes a critical obstacle to predict an application in tissue engineering. It remains unknown whether MSCs differentiation could be realized when the cells are cultivated on a scaffold that could be used in vascular engineering. In this study, we propose to differentiate human MSCs into endothelial-like cells on surfaces coated with polyelectrolyte multilayer film (PMF) and fibronectin (control surfaces). We quantified Platelet Endothelial Cell Adhesion Molecule (PECAM) and von Willebrand Factor (vWF) expressions (endothelial cell specific markers) and nitric oxide (NO) production, which is representative of the cell functionality. After only two weeks of differentiation, we showed, on PMF, that MSCs expressed PECAM and vWF, exhibiting a differentiation into endothelial-like cells, which functionality was explored by a significant production of nitrites. These results highlight the importance of PMF to get human MSCs differentiation and suggest that this film of nanometer thickness opens a new route for vascular bioengineering by pre-seeding hMSCs directly into a vascular graft functionalized by a removable coating. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.