Embryonic stem (ES) cells are pluripotent cells with indefinite replication potential and ability to differentiate into all types of cells. An understanding of the regulatory mechanisms responsible for pluripotency in ES cells is critical for realizing their potential in regenerative medicine and science. Cross-species studies on ES cells have identified pathways and networks that are either fundamental to or species-specific for self-renewal and differentiation. Although pluripotency as an essential function in multicellular organisms is conserved through evolution, mechanisms primed for differentiation contribute substantially to the differences among stem cells derived from different tissues or species. Transcriptome mapping analysis has determined the chromosomal domains of gene coexpression patterns specific to the ES state and demonstrated that regulation of ES cell development is operative at both the local chromosomal domain level and global level. Combinatorial signals from multiple pathways regulate the expression of key intrinsic factors critical for ES cell fate determination. The regulatory core formed by Oct4, Sox2, and Nanog, in particular, activates genes critical for self-renewal and represses genes initiating differentiation, controlling ES cell pluripotency. Here, we review recent findings on mechanisms controlling ES cell development. By integrating data from different sources, we present a global picture of how ES cells reach the decision of self-renewal or differentiation.