In this study we investigated the effects of biochemical reagents, direct co-culturing as well as flowinduced shear stress on the differentiation of MSCs into vascular cell lineages. To investigate the effects of growth factors on the differentiation of MSCs, medium with/without growth factors were used. To induce shear stress by fluid-flow on the cells, miniature flow cell chips were fabricated using polydimethylsiloxane. For the co-cultures, MSCs were mixed with HUVECs in the ratio of 1:2. 48h after the seeding, the shear stress was imposed on the cells. The flow rate was adjusted to have the shear stress as 10 dyne/cm2. Immunofluorescence stainings for α-smooth muscle actin (α-SMA) and von Willebrand factor (vWF) were performed. Acquired stained samples from a confocal microscope were analyzed by image processing. Any expressions of vascular specific markers were not shown in MSCs co-cultured with HUVECs when growth factors were not added. The expressions of α-SMA were observed in MSCs co-cultured with growth factor under static culture on day 4, but these were remarkably decreased on day 7. The expressions of α-SMA in MSCs co-cultured under shear stress were increased regardless of the growth factor. The highest expression of α-SMA was observed in MSCs co-cultured with flow-induced shear stress. The expressions of vWF were not observed in MSCs in all groups. In summary, we have shown that coculturing of MSCs and HUVECs under shear stressing, which mimics micro-environments, could help MSCs differentiate toward smooth muscle like cells.