Abstract: Neonatal calf male germ stem cells (mGSCs) and calf sertoli cells(CSCS) were separated and purified through the method of adhering velocity difference, and in vitro cultivation of mGSCs was studied on feeder layer cells of CSCS. The feeder layer cells of CSCs survived time did not exceed 8 d. Like-ES clusters formed when mGSCs was cultured on CSCS till 5~6 d. The method of mechanical dispersion could favor formation and presence of clusters. At lest in first 2 passages, this clusters existed. Cells of part clusters center were AKP positive staining and cells of clusters margin were AKP light positive or negative staining. Immunohistochemical analysis of typical clusters showed SSEA1 strongly positive, SSEA3 light positive and Oct-4 positive. The results suggested neonatal calf mGSCss can form ES cells.