ES cells maintain their self-renewal and pluripotency via a number of interrelated regulatory networks. The key to understanding development lies in dissecting the underlying crosstalk between these networks that function cooperatively to promote self-renewal and pluripotency. Our laboratory focuses on the role of two key transcription factors, Sox2 and Oct4 in the self-renewal of ES cells. Sox2 and Oct4 reside at the top of a critical gene regulatory network that is essential for embryogenesis. Sox2 and Oct4 regulate their target genes by binding to closely spaced HMG and POU cis regulatory elements respectively, which are located within the regulatory regions of their target genes. Recent work in our laboratory demonstrated that Sox2 levels need to be maintained at a precise level for ES cells to self-renew. Indeed, mouse ES cells engineered to inducibly overexpress Sox2 by just 2-fold cause the cells to differentiate. Several cell lineage-specific developmental regulators, including Sox21, are turned on under these conditions. The purpose of this dissertation is two-fold: (1) To identify novel regulators of ES self-renewal under the control of Sox2 and Oct4, and (2) To dissect the mechanisms underlying the regulation of a key developmental regulator, Sox21, during ES cell differentiation. We developed a three-step strategy for the rapid identification of a large number of putative Sox2:Oct4 target genes in ES cells, and demonstrate that DPPA4 (Developmental Pluripotency associated gene 4) is a bona tide Sox2:Oct4 target gene. We further demonstrate that a bivalent developmental regulator, Sox21, is bound by an array of activating and repressive machinery in ES cells, including Sox2 and Oct4. Upon the induction of differentiation by elevating Sox2 levels, we showed that the majority of the identified repressive complexes exit from the gene, allowing its rapid activation by the activating machinery. Moreover, we demonstrated that ES cells tightly regulate critical lineage-specific genes such as Sox21 to prevent its inappropriate activation, due to the presence of multiple redundantly functioning layers of repressive machinery. Taken together, the work presented in this dissertation provide significant new insights into the transcriptional control mechanisms utilized by ES cells to control self-renewal and pluripotency.