X-inactivation is a fundamental process of mammalian development, leading to the transcriptional silencing of one of the two X chromosomes in female cells. Studies of the X-inactivation process in the human system have been limited due to the lack of a cellular system that faithfully recapitulates the initiation of X-inactivation. In the mouse, ESCs faithfully recapitulate the initiation of X-inactivation and induce expression of the long noncoding RNA Xist, the major regulator of X-inactivation, on one of the two X chromosomes upon differentiation, leading to the silencing of the chromosome. Thus, mouse ESCs carry two active X chromosomes (Xa’s) and gain an XIST RNA-coated inactive X (the Xi) upon differentiation. Contrary, conventionally cultured human pluripotent stem cells (hPSCs) (both ESCs and iPSCs) do not display a stable XaXa state that allows silencing of the X chromosome via induction of XIST upon induction of differentiation. Instead, these cells display varying epigenetic states of the X chromosome, predominantly carrying an inactive X (Xi) with XIST expression, an Xi without XIST expression, or a partially reactivated Xi, the eroded Xe, and even change their X state over time in culture. Furthermore, none of the X-states found in human PSCs resembles that of the human blastocyst (XaXa with both chromosomes expressing XIST). Therefore, typical human PSCs are in a developmental state that endows an epigenetic instability on the X chromosome, and is past the early developmental window that allows for de novo initiation of X-inactivation. Given the importance of X-inactivation for development and its close ties to pluripotency, surprisingly little is known about how X-inactivation is initiated in the human system. To overcome this gap in knowledge, in this proposal, we established and characterized a pluripotent cell culture system that resembles the blastocyst-like pattern of the X chromosome. With this tool in hand, we are studying the mechanisms of X-inactivation during human development, and are addressing how XIST function in human cells differs from that in the mouse. In addition, we are testing whether these cells are epigenetically better suited for both basic scientific and translational goals than hPSCs in grown under conventional culture conditions.