Normal somatic cells possess a finite life span owing to replicative senescence. Telomerase functions as a potential regulator of senescence in various cells. Expression level of human telomerase reverse transcriptase (hTERT) is correlated with telomerase activity and cellular immortalization. In this study, we investigated the effects of ectopic expression of hTERT on proliferation potential of chicken feather keratinocyte stem cells (FKSCs). We established FKSCs transduced with hTERT catalytic subunit fused with EGFP marker gene (hTERT-EGFP-FKSCs). hTERT-EGFP-FKSCs had the great potential of proliferation in vitro and expressed kerainocyte stem cell markers integrin beta1 and CD49c. Keratin 15 and keratin 19, as native FKSCs, were also detected in hTERT-EGFP-FKSCs. By the analysis of fluorescent RT-PCR, western blotting and TRAP assay, hTERT-EGFP-FKSCs were positive for telomerase activity, in comparison with native FKSCs showing no telomerase activity. We demonstrated that ectopic expression of hTERT could result in immortalization of FKSCs. Tumorigenecity of hTERT-EGFP-FKSCs were examined by soft agar assay and transplantation into NOD-SCID mice. Results showed that hTERT-EGFP-FKSCs sustained the cellular characteristics of native FKSCs and had no transforming activity. In vivo differentiation multipotentials of hTERT-EGFP-FKSCs were confirmed by transplantation into developing chicken embryos and in situ hybridization analysis. These data provide a novel framework for understanding human telomerase activity in different species and suggest a new insight for manipulating hTERT for therapeutic purposes in treating tissue injury and aging.