Designing convenient substrates is a pertinent parameter that can guide stem cell differentiation. Current research is directed toward differentiating mesenchymal stem cells (MSCs) into endothelial cells (ECs). It is generally accepted that MSCs cannot be easily differentiated into ECs without high concentrations of proangiogenic factors. To guide either bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and Wharton's jelly‐derived mesenchymal stem cells (WJ‐MSCs) into ECs‐like phenotype, poly(allylamine‐hydrochloride)/poly(styrene‐sulfonate) multilayers film (PAH/PSS) was used as culture coating and compared to type I collagen (as control coating). After 2 weeks of culture and in absence of angiogenic growth factors, PAH/PSS upregulated KDR, PECAM‐1, and CDH5 genes, whereas combining PAH/PSS with endothelial growth media (EGM‐2^®) led to the production of respective proteins by WJ‐MSCs. In contrast, not fully EC‐like phenotype is obtained from the differentiation of BM‐ or WJ‐MSCs cultured on type I collagen. Thus, using PAH/PSS coating in synergy with EGM‐2^® appears as an ideal condition promoting WJ‐MSCs differentiation into ECs‐like. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 292–300, 2017.