In adult mammalian brain, microvessels may produce a special microenvironment and then influence the characteristics of neural stem cells (NSCs). This work aims to compare the effects of cerebral microvascular endothelial cells (CMECs) and vascular endothelial growth factor (VEGF) on the behaviors of NSCs. An in vitro model with co-cultured CMECs and NSCs was established by using transwell co-culture system. The expression of nestin and NF in the early stage of the co-culture, and NF in the later stage were explored by immunostaining. Results demonstrate that both CMECs and VEGF stimulate the proliferation of NSCs and inhibit the differentiation. After removing CMECs or VEGF from the co-culture system, a further culture in medium for the differentiation of NSCs was performed. The percentage of NF-positive cells in the CMECs co-culture group is the highest, suggesting the improved neuronal differentiation of NSCs by CMECs.