Objective:To establish C57BL/6J mouse embryonic stem (ES) cell lines with bFGF-free knockout serum replacement (KSR). Methods:C57BL/6J mouse blastocysts 3.5 days post coition(dpc) were collected and cultured in the medium supplemented with KSR and 1 000 U/mL LIF, with or without 10 ng/mL bFGF. The ES cell lines were identificated by morphology, alkaline phosphatase activity, karyotype, cell surface maker SSEA-1 and pluripotent markers (Oct4, Sox2 and Nanog) analysis. The differentiation ability of ES cells was analyzed both in vitro (embryoid bodies) and in vivo (teratoma). Results:Four cell lines were established out of 6 blastocysts cultured in the medium without bFGF; in contrast, no ES-like colony was observed when bFGF was added to the medium after isolation of 3 high quality blastocysts. The ES cells proliferated well without evidence of differentiation. The ES cells had typical ES-like morphology, expressed alkaline phosphatase,SSEA-1, pluripotent makers such as Oct4, Sox2 and Nanog. They could form embryoid bodies when been cultured in suspension and generate teratomas when been injected subcutaneously into SCID mice, consistent with the the pluripotent abilities of ES cells. Conclusion:KSR containing medium without bFGF could be an efficient culture system for the establishment of mouse ES cell lines.