Embryonic stem cells (ESCs) possess unlimited self-renewal capacity and developmental potential to differentiate into virtually any cell type of an organism.In recent years, proteomic approaches have produced a wealth of data identifying proteins and mechanisms involved in ESCs proliferation and differentiation.ESC lines contain relatively abundant nuclear proteins in terms of both variety and protein content.In total, ninety-two proteins were commonly identified in both mouse and human ESCs.Nowadays, two-dimensional gel electrophoresis(2-DE),Mass spectrometry(MS),MS-based protein profiling and quantitative analysis, and protein chips approach have been applied to ESCs research.However, further optimization of these techniques is needed before they can be widely used in proteome analyses.The following review summarizes the methodologies used with proteome analyses of ESCs, and discusses biological and technical challenges for ESCs proteomics research.