Major complications with in vitro culture of female embryonic stem cells (mESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female mESC system that has enabled us to produce an optimised protocol for preserving the XX karyotype; a protocol that also improves male mESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation. This is the first screen that has studied the establishment of X inactivation in its native context, again based on the challenges of female mESC culture. We reveal a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. To the best of our knowledge this hasn't been reported previously. Our female mESC system provides a tractable model for XX mESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation.