Human embryonic stem cells (hESC) have great potential in regenerative medicine, provided that culture systems are established that maintain genomic integrity. Here we describe a comparison of the effects of culture in either physiologic oxygen (2%) or room oxygen (21%) on the hESC lines, H1, H9, and RH1. Physiologic oxygen enabled an average sixfold increase in clone recovery across the hESC lines tested (p <0.001). FACS analysis showed that cells cultured in physiologic oxygen were significantly smaller and less granular. No significant changes had occurred in levels of SSEA4, SSEA1, TRA-1-60, or TRA-1-81. While karyotypic normalcy was maintained in both H1 and H9, the frequency of spontaneous chromosomal aberrations was significantly increased in room oxygen. This increase was not observed in physiologic oxygen. These results clearly demonstrate that physiologic oxygen culture conditions are indispensable for robust hES clone recovery and may enhance the isolation of novel hES lines and transgenic clones.