The presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency specific to murine embryonic stem cells (ESCs). Human ESCs invariably exhibit signs of X chromosome inactivation (XCI) and are considered developmentally more advanced than their murine counterparts. We describe the establishment of the first XaXa hESCs derived under physiological oxygen concentrations. Using these cell lines we demonstrate that (1) differentiation of hESCs induces random XCI in a manner similar to murine ESCs, (2) chronic exposure to atmospheric oxygen is sufficient to induce irreversible XCI with minor changes of the transcriptome, (3) the Xa exhibits heavy methylation of the XIST promoter region, and (4) XCI is associated with demethylation and transcriptional activation of XIST along with H3K27-me3 deposition across the Xi. These findings indicate that the human blastocyst contains pre-X-inactivation cells and that this state is preserved in vitro through culture under physiological oxygen. Examination of 2 different histone modifications (H3K4me3 and H3K27me3) in 3 ES cell lines in 5% and 20% oxygen.