Induction of autologous stem cells for directed differentiation has become a predominant method to obtain autologous cells for tissue reconstruction. However, the low inducing efficiency and contamination with other type of cells hinder its clinical utilization. Here we report a novel phenomenon that the corneal epithelial cells maintain long-term proliferative capacity and tissue-specific cell phenotype by factors secreted from murine embryonic stem cells (ESCs). The rabbit corneal epithelial cells grew very well in culture medium with addition of 40% ESC conditioned medium (ESC-CM). These corneal epithelial cells have been serially subcultured for more than 20 passages and maintained high cell purity, cobble-stone-like morphology, enhanced colony forming efficiency, normal diploid, and capacity to regenerate a functional stratified corneal epithelial equivalent. More importantly, these cells did not form tumor, and the cells lost their proliferative capacity after withdrawal of ESC-CM. The long-term proliferative capacity of corneal epithelial cells is partly resulted from enhancement of cell survival and colony formation, and mediated by ectopic expression of telomerase. Our findings indicate that this new ESC-CM culture system can generate low-immunogenic autologous cells sufficiently for use in regenerative medicine.