Abstract: The chicken embryos incubated for 8?12 days were trypsinized to dissociate the chicken embryonic fibroblasts （CEF）. CEF were maintained in DMEM containing 4.5 g/L glucose，10% newborn calf serum（NBS） as the feeder layer for culturing chick-en ES cells after treated with 10 mg/L Mitomycin C. In order to define the growth re-quirement for proliferation of ES cells,the sera and the chicken embryonic extract were screened by the methods of testing CEF’s dehydrogenase activity, and the chicken ES cell’s optimal culturing system which composed of DMEM （high glucose） containing 10%NCS，5%FBS，2.5% chicken embryo extract, 10μg/L human bFGF，1×10⁶/L mLIF，40μg/L Conalbumin，and 0.14mmol β-Mercaptoethanol （β-ME） were estab-lished.The alkaline-phosphatase-positive （chicken ES ） cells growing well and main-tained undifferentiated until 9 passages in the feeder layers of the optimal culturing sys-tem.