Background: Human pluripotent stem cells (hPSCs) are de-fined by their ability to differentiate into derivatives of three embryonic germ layers and displayed two distinct states of pluripotency; naïve and primed. Naïve hPSCs resemble to pre-implantation epiblast cells and stand for a higher developmental potential while primed hPSCs correspond to post-implantation epiblast and primed to differentiation. The chimera formation is considered as one of the most stringent assay for evaluation of pluripotency in naïve and primed hPSCs; however, this strat-egy is challengeable for human cells due to the ethical issues. As an alternative, interspecies chimera formation between hP-SCs and an appropriate animal embryo can be a solution. Due to the developmental similarities in the gastrula-stage of chick and human embryos, we assumed that the chick embryo might be a suitable host for receiving hPSCs in interspecies chimera formation.Materials and Methods: In this study, we compared the ef-ficiency of interspecies chimerism for naïve and primed hPSCs after injection into the chick embryos at the blastoderm and primitive streak stages, respectively, in the developmental stage matching and non-stage matching approaches. After eight days of injection, the presence of hPSCs’s derivatives were traced by GFP detection and immune staining for HNA and three germ layer specific markers also further verified with a sensitive genomic Alu PCR assay.Results: In the matched developmental stage approach we showed the high ability of both naïve and primed states in par-ticipating and differentiating in the developing host embryos. More importantly, we showed that, chick embryos could pro-vide an opportunity for evaluating the hPSCs’s pluripotency when they injected into the host embryos as non-developmental stage match approach by survival, proliferation and participa-tion in the developing chick embryos.Conclusion: Chick embryo can be a suitable host for evaluat-ing the hPSCs pluripotency states and provide provocative ap-proach for in vivo differentiation as well as human organoid formation during blastoderm complementation