Adult stem cell-derived smooth muscle cells (SMC) may be a promising source of cells for applications in regenerative medicine, including cardiovascular tissue engineering. Primary SMC from native vessels may have limited proliferative capacity and reduced collagen production when sourced from elderly donors, who are the patients in need of vascular grafts due to coronary disease or peripheral arterial disease. Our recent work showed that the ability of human bone marrow-derived mesenchymal stem cells (hMSCs) to differentiate into SMC was modulated by various growth factors, matrix proteins, and mechanical forces. In addition, the components of the culture medium play a very important role in SMC differentiation from hMSCs. In this chapter, we will summarize our experience with the impact of various factors on SMC differentiation from hMSCs. Based upon our findings regarding growth factors, cyclic strain and matrix proteins, a two-phase vessel regeneration culture protocol including a 4-week proliferation phase and a 4-week differentiation phase was developed to optimize proliferation and SMC differentiation of hMSCs consecutively.