Abstract: Objective To investigate the characteristics of differentiation of lung side population cells (LSP cells)in vitro. Methods CD45-/CD31+ LSP cells sorted by flow cytometry were taken from mouse lung tissues and cultured for 14 d. The cultured LSP cells were observed with colony formation assay and flow cytometryin vitro. The mRNA expressions of ATP-binding cassette transporter G2 (ABCG2), smooth muscle actin (SMA) and α-smooth muscle tropomyosin (α-SMT) in both freshly isolated LSP cells and cultured LSP cells were examined. The expressions of ABCG2 and stem cell antigen 1 (Sca1) in LSP cells were detected using immunofluorescence. RT-PCR tests were performed to detect the expressions of ABCG2, SMA and α-SMT in LSP cells. Results The isolated CD45-/CD31+ lung side population cells expressed ABCG2, SMA and Sca1, but not α-SMT. A large number of LSP in aggregated state were observed after 14 d of culture. Before induction of differentiation, the CD45-/CD31+ LSP cells expressed ABCG2 and SMA, but not α-SMT. After induction of differentiation, the CD45-/CD31+ lung side population cells expressed α-SMT and SMA, but not ABCG2. Conclusion CD45-/CD31+ LSP cells might be progenitor cells of vascular smooth muscle cells，possessing the characteristics of stem cell differentiations.