The study of ES cell-mediated neuronal differentiation allows elucidating the mechanism of neuronal development in spite of the complexity and the difficult accessibility. During the differentiation of embryonic stem cells into neuronal cell, the expression profiles in the level of protein were extensively investigated by proteomic analysis. These cells were analyzed for charges in proteome during the differentiation of ES cells by 2-dimensional electrophoresis (2-DE) and MALDI-TOF MS. Seven unique proteins were identified, some of which were differentially expressed at each stage. A complex system of neuronal differentiation can be activated in cultured embryonic stem cells and our two dimensional electrophoresis data should be useful for investigating some of the mechanism that regulates neuronal differentiation.