Our understanding of self-renewal and differentiation capacity of human embryonic stem cells (hESCs) remains elusive on the detailed molecular mechanisms. Our current study elucidates the global regulation of stem cell beyond the well-known stem cell factors by combining over 33 microarrays and the latest bioinformatic tools. We examined if there are a set of key genes consistently altered during differentiation of hESCs regardless of differentiation conditions. By comprehensive genome-wide consensus microarray analyses, we have profiled gene expression signatures that are most significantly affected by differentiation in hESCs from our own microarray data sets as well as publically available microarrays. Our finding has unveiled the novel molecular markers that determine self-renewal and form intramodular hubs. Bioinformatics approach for the identification of new molecular markers defining undifferentiated hESCs, interacting partners and interconnectivity analyses may contribute to delineating molecular mechanisms of stem cell self-renewal/differentiation and can be a useful tool to identify molecular factors inducing stemness from different cell types.