Embryonic stem (ES) cells are totipotent stem cells, which can differentiate into various kinds of cell types, including neurons. They are widely used as a model system for investigating mechanisms of differentiation events during early mouse development. In this study, proteomic techniques were used to approach the protein profile associated with the early-stage differentiation of ES cells into neuronal cells induced by all-trans retinoic acid (ATRA) in vitro. In comparison of the protein profile of parent ES cells with that of ES-derived neural-committed cells, which was induced by ATRA for four days, 24 differentially displayed protein spots were selected from two-dimensional electrophoresis (2-DE) gels for further protein identification by pepide mass fingerprinting (PMF). Nine proteins were known to being involved in the process of neural differentiation and/or neural survival. Of those, alpha-3/alpha-7 tubulin and vimentin were down-regulated, while cytokeratin 8, cytokeratin 18, G1/S-special cyclin D2, follistatin-related protein, NEL protein, platelet-activating factor acetylhydrolase IB alpha-subunit, and thioredoxin peroxidase 2 were upregulated during differentiation of ES cells to neural cells. Additionally, other 12 protein (five upregulated and seven downregulated) spots associated with ES cell differentiation into neuronal cells were not matched to known proteins so far, implicating that they might be novel proteins. The results above indicated that the molecular mechanisms of differentiation of ES cells to neural cells in vitro might be similar to those of other neural systems in vitro and identified that proteomic analysis is an effective strategy to comprehensively unravel the regulatory network of differentiation.