During the last 5 years there has been enormous progress in developing a deeper understanding of the molecular mechanisms that control the self-renewal and pluripotency of embryonic stem cells (ESC). Early progress resulted from studying individual transcription factors and signaling pathways. Unexpectedly, these studies demonstrated that small changes in the levels of master regulators, such as Oct4 and Sox2, promote the differentiation of ESC. More recently, impressive progress has been made using technologies that provide a global view of the signaling pathways and the gene regulatory networks that control the fate of ESC. This review provides an overview of the progress made using several different high-throughput technologies and focuses on proteomic studies, which provide the first glimpse of the protein–protein interaction networks used by ESC. The latter studies indicate that transcription factors required for the self-renewal of ESC are part of a large, highly integrated protein–protein interaction landscape, which helps explain why the levels of master regulators need to be regulated precisely in ESC. J. Cell. Physiol. 227: 27–34, 2012. © 2011 Wiley Periodicals, Inc.