ABSTRACT A major goal of stem cell research is to derive high-quality human pluripotent stem cells (hPSCs) that possess the unique transcriptional, epigenetic, and functional properties of developmentally unrestricted “naïve” pluripotent cells found in the pre-implantation blastocyst in vivo. In contrast, hPSCs derived under traditional conditions represent a more advanced or “primed” developmental state that corresponds to the late post-implantation epiblast. My work and that of others has shown that primed hPSCs can be reprogrammed into a naïve state marked by globally reduced DNA methylation levels, X chromosome reactivation in female cells, and a broad capacity for differentiation along extraembryonic fates. However, the widespread utilization of naïve hPSCs in biomedical research is currently impaired by epigenetic instability during establishment and maintenance of the naïve state. In particular, naïve hPSCs generated with currently available methods display irreversible erasure of parent-specific imprinting, retain an epigenetic memory of the inactive X chromosome in female cells, and are not directly responsive to lineage cues for embryonic germ layer induction. My long-term goal is to design rational strategies to derive naïve hPSCs that faithfully recapitulate pluripotent cells in vivo and establish a robust cellular platform for modeling human development and disease. Based on our published and preliminary data, I hypothesize that aberrant epigenetic reprogramming during induction of naïve pluripotency is a consequence of inappropriate signal perturbation. I propose to enhance the epigenetic fidelity of naïve hPSCs by pursuing three complementary research directions. The first is to elucidate the signaling cascade during early stages of the primed-to-naïve transition using quantitative phosphoproteomics, as a basis for developing strategies to activate the naïve transcriptional circuitry without inadvertently inducing genetic or epigenetic instability. Second, I will employ recently established fluorescent reporter cell lines in combination with chemical and genetic approaches to preserve parent-specific epigenetic information during the establishment of naïve pluripotency and promote random XCI upon exit from naïve human pluripotency. Third, I will enhance the competence of naïve hPSCs for rapid induction of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) by targeted epigenome editing of cis-regulatory elements that gain accessibility during the transition from naïve to primed pluripotency. The overall aim of this project is to exploit the presently untapped biomedical potential of naïve hPSCs and generate a robust in vitro platform to model early human development and disease.