Many fundamental issues related to the regulation of stem cell function and fate, especially with regard to human cells, remain to be resolved. X chromosome inactivation (XCI or X-inactivation) is one of those fundamental processes of human development related to stem cell biology, that we know surprisingly little about. To be equal with males, females shut off one of two X chromosomes during development by inducing X-inactivation, such that only one X chromosome remains active in every cell of the human female body. XCI is an epigenetic phenomenon that occurs without alterations in the primary sequence of DNA but instead by formation of a repressive heterochromatin structure on an entire X chromosome. Studies on X-inactivation in the mouse model system have revealed that female embryonic stem cells (ESCs) carry two active X’s and that the X-inactivation process is initiated when these cells differentiate such that all differentiated cells have an inactive X chromosome. Intriguingly, the heterochromatin structure of the inactive X chromosome can be erased when adult cells of the mouse are reprogrammed to the mouse ESC-like pluripotent state also coined induced pluripotency. Notably, the X-inactivation process is much less studied in the human system. Furthermore, some of the existing data already suggest that the process is regulated differently in human than in mouse. Therefore, in this project, our goal is to begin to understand how XCI is initiated in the human system. Towards this end, we are working towards the establishment of a pluripotent stem cell model that allows us to follow the initiation of XCI in a cell culture model. We are proposing that the identification of such pre-XCI human pluripotent cells, which undergo X-inactivation upon differentiation, has implications for the use of human pluripotent cells in therapeutic applications, modeling of human development and diseases, as the XCI status affects all these applications. During the past funding period, we have tested various approaches for the generation of pre-XCI human pluripotent cells and are currently further refining these methods. Along this line, we established tools for better identifying the pre-XCI human pluripotent cells and studying the initiation of X-inactivation. In addition, we made considerable progress in defining the epigenetic instability of the inactive X in certain pluripotent human stem cell lines.