Proteome analyses of embryonic stem cells (ESCs) will help to uncover mechanisms underlying cellular differentiation, expansion, and self-renewal. We applied a 2-DE based proteomic approach coupled with mass spectrometry to identify genes controlling monkey ESCs proliferation and differentiation. We analyzed proteome of ESCs during proliferation and different stages of spontaneous differentiation (day 3, 6, 12, and 30) by embryoid body formation. Out of about 663 ± 15 protein spots reproducible detected on gels, 127 proteins showed significant changes during differentiation. Mass spectrometry analysis of differentially expressed proteins resulted in identification of 95 proteins involved in cell cycle progression and proliferation, cell growth, transcription and chromatin remodeling, translation, metabolism, energy production and Ras signaling. In addition, we created protein interaction maps and distinctly different topology was observed in the protein interaction maps of the monkey ESC proteome clusters compared with maps created using randomly generated sets of proteins. Taken together, the results presented here revealed novel key proteins and pathways that are active during ESC differentiation.