The nuclear proteome enables, manages, and regulates the genome by the collective actions and interactions of proteins found in the nucleus. We applied a proteomic approach to analyze a nuclear proteome during embryonic stem cell (ESC) proliferation, and 3 and 9 days after initiation of differentiation. The nuclei were isolated from cells and their proteins were separated using 2-DE. Out of about 560 protein spots reproducible detected on any give gel, 49 differentially expressed proteins were identified by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI TOF/TOF) mass spectrometry. Of them, several nuclear located proteins involved in chromatin remodeling, transcription regulation, apoptosis, cell proliferation, and differentiation were identified including CTBP1, MM-1, RUVBL1, HCC-1, SGTA, SUMO2, and Galectin-1. Functional interaction analysis of differentially expressed proteins revealed that most of nuclear proteins had a direct interaction with c-Myc and p53.