Objective:research systematically into the isolation,culture and direct differentiation to osteoblast of embryonic stem cells.  Method: To obtain feeder layer of primary mouse embryo fibroblasts(PMEF), 13.5d pregnant KM mouse was chosen as a donor. After using mitomycin-C treated PMEF, they were used as feeder cells in media of DMEM for isolation ES cell colonies and for coculture with ES-D3 cells. We flushed blastocysts from pregnant uterus at p.c. 4.After 4 to 5 days,nest-like clones of inner cell mass appeared and were dispersed by 0.25% trypsine-0.02