On the basis of our previous report verifying that chemokine (C-X-C motif) receptor 2 (CXCR2) ligands in human placenta-derived cell conditioned medium (hPCCM) support human pluripotent stem cell (hPSC) propagation without exogenous basic fibroblast growth factor (bFGF), this study was designed to identify the effect of CXCR2 manipulation on the fate of hPSCs and the underlying mechanism, which had not been previously determined. We observed that CXCR2 inhibition in hPSCs induces predominant differentiation to mesoderm and endoderm with concomitant loss of hPSC characteristics and accompanying decreased expression of mammalian target of rapamycin (mTOR), β-catenin, and human telomerase reverse transcriptase (hTERT). These phenomena are recapitulated in hPSCs propagated in conventional culture conditions, including bFGF as well as those in hPCCM without exogenous bFGF, suggesting that the action of CXCR2 on hPSCs might not be associated with a bFGF-related mechanism. In addition, the specific CXCR2 ligand growth-related oncogene α (GROα) markedly increased the expression of ectodermal markers in differentiation-committed embryoid bodies derived from hPSCs. This finding suggests that CXCR2 inhibition in hPSCs prohibits the propagation of hPSCs and leads to predominant differentiation to mesoderm and endoderm owing to the blockage of ectodermal differentiation. Taken together, our results indicate that CXCR2 preferentially supports the maintenance of hPSC characteristics as well as facilitates ectodermal differentiation after the commitment to differentiation, and the mechanism might be associated with mTOR, β-catenin, and hTERT activities.