A defining feature of female pluripotent mouse embryonic stem cells (mESCs) is the bi-allelic gene dosage from two transcriptionally active X chromosomes, as opposed to the single dosage in XY males. X chromosome gene dosage is typically equalised between XX females and XY males upon differentiation through chromosome-wide transcriptional suppression in a process known as X chromosome inactivation (XCI). In vitro female mESCs do not display XCI and are instead in a pre-inactivated state with both X chromosomes transcriptionally active. The pre-inactivated state is very transient in vivo, however, in vitro female mESCs, unable to undergo XCI due to restrictive media, instead, rapidly lose one of the two X chromosomes to achieve dosage compensation, adopting an XO karyotype. XO female mESCs lose the defining features of female-specific pluripotency and instead resemble their XY male counterparts epigenetically, morphologically, and functionally, rendering these cells unusable for female-specific pluripotency research. To use XX female mESCs and their in vitro derived equivalent human iPSCs for research and regenerative therapies, the genes which regulate XX karyotype stability and XCI in ESCs need to be identified. Here we suggest that a whole-genome CRISPR knock-out screen in Xmas mESCs, which express fluorescent reporters for X chromosome karyotype status, will identify regulators of female pluripotency, differentiation, and XCI.