Gene expression analyses of the embryonic stem cells (ESCs) will lead to uncover or further define signaling pathways and molecular mechanisms involved in the maintenance of self-renewal and pluripotency. We performed a mass spectrometry-based quantitative analysis using 8-plex iTRAQ labeling to identify differentially expressed proteins in human ESC line, Royan H5, during spontaneous differentiation by embryoid body formation in different stages (ESC, EBs 6, 12 and 20 days after differentiation initiation). Evaluated by strict criteria, we successfully identified 391 proteins present in more than three replicates, including different proteins involved in cell proliferation, cell apoptosis, transcription, translation, mRNA processing and protein folding. The differential expression pattern was significantly observed among 190 proteins such as down-regulation of important proteins like Lin- 28 homolog A, BAF155, and Galectin-1 which are taking part in regulating pluripotency and proliferation in ESCs as well as up-regulation of various proteins such as Ezrin, Thrombospondin-1, Serpin B9, Protein DJ-1 and Cathepsin B. The further analysis is in progress.