Human parthenogenetic embryonic stem (pES) cells can be clinically used in the future to avoid immunological rejection. However, the developmental potential of human pES cells remains to be elucidated. In this study, we generated a human pES-enhanced green fluorescent protein (EGFP) cell line (chHES-32-EGFP), which shows pluripotency thus far and maintains stable and robust EGFP expression in the undifferentiated and differentiated states in vivo and in vitro. Using this pES-EGFP cell line, we found that when human pES-EGFP cells were injected into mice blastocysts, EGFP-positive cells progressively decreased with the development of blastocysts in vitro. Only 4 out of 23 embryos (17.4%) contained EGFP-positive cells and all of these embryos exhibited abnormal morphology or delayed development when the chimera blastocysts were implanted into the pseudopregnant recipient mouse uterus. These results raise serious questions regarding the feasibility of the generation of interspecific chimeras between mouse blastocysts and human pES cells.