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Genetic and epigenetic alterations are observed in long‐term culture (&gt;30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large‐scale gene expression analysis between initial‐passage hESCs (ihESCs, &lt;10 passages) and early‐passage hESCs (ehESCs, 20–30 passages) of 12 hESC lines, we found that the  DLK1‐DIO3  gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the  DLK1‐DIO3  active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the  DLK1‐DIO3  cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage‐induced apoptosis in ehESCs and their differentiated hepatocyte‐like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the  DLK1‐DIO3  cluster. Overall, the data suggest that active expression of the  DLK1‐DIO3  cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs. S tem  C ells   2014;32:391–401

Physiological Oxygen Prevents Frequent Silencing of the DLK1‐DIO3 Cluster during Human Embryonic Stem Cells Culture