Four commercially available serum-free and defined culture media tested on 2 human embryonic stem cell (hESC) lines were all found to support undifferentiated growth for >10 continuous passages. For hESC cultured with defined StemPro and mTeSR1 media, the cells were maintained feeder-free on culture dishes coated with extracellular matrices (ECMs) with no requirement of feeder-conditioned media (CM). For xeno-free serum replacer (XSR), HEScGRO, and KnockOut media, mitotically inactivated human foreskin feeders (hFFs) were required for hESC growth. Under the different media conditions, cells continued to exhibit alkaline phosphatase activity and expressed undifferentiated hESC markers Oct-4, stage-specific embryonic antigens 4 (SSEA-4), and Tra-1-60. In addition, hESC maintained the expression of podocalyxin-like protein-1 (PODXL), an antigen recently reported in another study to be present in undifferentiated hESC. The cytotoxic antibody mAb 84 binds via PODXL expressed on hESC surface and kills >90% of hESC within 45 min of incubation. When these cells were spontaneously differentiated to form embryoid bodies, derivatives representing the 3 germ layers were obtained. Injection of hESC into animal models resulted in teratomas and the formation of tissue types indicative of ectodermal, endodermal, and mesodermal lineages were observed. Our data also suggested that StemPro and mTeSR1 media were more optimal for hESC proliferation compared to cells grown on CM because the growth rate of hESC increased by 30%-40%, higher split ratio was thus required for weekly passaging. This is advantageous for the large-scale cultivation of hESC required in clinical applications.