Objective- This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells, and to examine the functional status of the newly differentiated endothelial cells. Methods and Results- Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells, and induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, NOS activity and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression re-occurred at day 7 during differentiation. Increasing eNOS mRNA, protein content and activity were observed in the cells at day 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal regulated kinas (ERK)₁ and ERK₂ occurred in these cells, which was inhibited by NOS inhibitor N-nitro-L-arginine methyl ester. Conclusion- These data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.