To realize the full potential of human embryonic stem cells (hESCs), it is important to develop culture conditions that maintain hESCs in a pluripotent, undifferentiated state. A low O(2) atmosphere (approximately 4% O(2)), for example, prevents spontaneous differentiation and supports self-renewal of hESCs. To identify genes whose expression is sensitive to O(2) conditions, microarray analysis was performed on RNA from hESCs that had been maintained under either 4% or 20% O(2). Of 149 genes differentially expressed, 42 were up-regulated and 107 down-regulated under 20% O(2). Several of the down-regulated genes are most likely under the control of hypoxia-inducing factors and include genes encoding enzymes involved in carbohydrate catabolism and cellular redox state. Although genes associated with pluripotency, including OCT4, SOX2, and NANOG were generally unaffected, some genes controlled by these transcription factors, including LEFTY2, showed lowered expression under 20% O(2), while a few genes implicated in lineage specification were up-regulated. Although the differences between O(2) conditions were generally subtle, they were observed in two different hESC lines and at different passage numbers. The data are consistent with the hypothesis that 4% O(2) favors the molecular mechanisms required for the maintenance of pluripotency.