Long-term in vitro maintenance of embryonic stem cell (ESC) pluripotency enables the pluripotency and differentiation of ESCs in animals to be investigated. The ability to successfully maintain and differentiate chicken embryonic stem cells (cESCs) would provide a useful tool for avian biology research and would be a resource directly applicable to agricultural production. In this study, endogenous chicken pluripotency transcription factors, POUV, Sox-2, Nanog and Lin28 were cloned and expressed as recombinant proteins containing a nine consecutive arginine protein transduction domain (PTD). cESCs were cultured with these recombinant proteins to maintain cESC pluripotency in vitro. Cultured cESCs exhibited typical characteristics of pluripotency, even after six generations of rapid doubling, including positive staining for stage-specific embryonic antigen I, and strong staining for alkaline phosphatase. Expression levels of the pluripotency markers, POUV, Nanog, C-Myc, Sox-2 and Lin28 were the same as in uncultured stage X blastoderm cells, and most significantly, the formation of embryoid bodies (EBs) by 6th generation cESCs confirmed the ability of these cultured cESCs to differentiate into cells of all three embryonic germ layers. Thus, transcription factors could be translocated through the cell membrane into the intracellular space of cESCs by using a PTD of nine consecutive arginines and the pluripotency of cESCs could be maintained in vitro for at least six generations.