Abstract: BRL-3A conditioned medium combined with basic Fibroblast Growth Factor (bFGF), Insulin-like Growth Factor (IGF), Stem Cell Factor (SCF) and STO cells are used for the culture of the feeder layer and ES (Embryonic Stem) cell clones. This study is done by isolation of chicken X-stage blastodermal cells. The putative ES cell clone features a typical bird’s nest structure, PAS and AKP-positive and strongly positive for SSEA-1. It is able to form embryoid bodies and multiple types of cells as well as participates in embryonic development and feather color chimera formation through in vitro differentiation.