Objective:To investigate the culture method for adult olfactory epithelium neural stem cells and their multipotency during in vitro differentiation.Methods: Olfactory epithelium was sequentially digested with neutral protease and collagenase ⅠA,then the olfactory epithelium neural stem cells were enriched and amplified.Neurospheres were formed by culturing the cells with serum free medium containing bFGF and EGF.Differentiation of neurospheres was induced with medium containing serum; the differentiated cells were identified by immunocytochemistry.Results: Primary cultured olfactory epithelium neural stem cells proliferated and formed colonies.Cells in the colonies expressed the in vivo markers GBC2 and cytokeratin 5.Passaged cells proliferated and formed neurospheres when cultured with serum free medium containing bFGF and EGF.Medium containing serum induced the differentiation of neurospheres.TUJ1 positive neurons,P75NGFR positive olfactory ensheathing cells,GFAP positive astrocytes and NG2 positive oligodendrocyte progenitors were detected in the highly heterogeneous differentiated cells; besides,the cells also contained nestin positive neural stem cells.Conclusion: Cultured adult olfactory epithelium neural stem cells can proliferate and form neurospheres.The neurosphere cells have the multipotency to differentiate into neurons,astrocytes and oligodendrocyte progenitors.