In placental mammals, dosage compensation occurs by silencing one X-chromosome in female cells, a process known as X-chromosome inactivation. Unlike female mouse embryonic stem cells (ESCs), which possess two active X chromosomes and undergo XCI upon induction of differentiation, female human ESCs exhibit various epigenetic states of the X chromosome, indicating a surprising epigenetic instability of these cells under normal culturing conditions. Since this epigenetic variation could have implications for the use of female human ESCs in regenerative medicine, disease studies, and basic research, in this proposal, we are aiming to determine how the epigenetic variability of the X chromosome arises during derivation and maintenance of human ESCs, the causes and consequences of deregulation of XCI in human ESCs, and to devise methods of stabilizing Xist expression in human ESCs. During the first funding period, we have extensively characterized the epigenetic state of the X chromosome in many established and newly derived human ESC lines as well as in human blastocysts. Together, our findings reveal new insights into the relationship between different X chromosome states in undifferentiated female human ESCs, clarify how they arise during ESC derivation, and define the implications of these X chromosome status for differentiated cells. The findings from our study have implications for the utilization and quality assessment of human ESCs.