The derivation of human embryonic stem cells (hESCs) brought cell therapy-based regenerative medicine significantly closer to clinical application. However, expansion of undifferentiated cells and their directed differentiation in vitro have proven difficult to control. This is mainly because of a lack of knowledge of the intracellular signaling events that direct these complex processes. Additionally, extracellular factors, either secreted by feeder cells that support self-renewal and maintain pluripotency or present in serum supplementing proprietary culture media, that influence hESC behavior are largely unknown. Xeno-free media that effectively support long-term hESC self-renewal and differentiation to specific types of specialized cells are only slowly becoming available. Microarray-based transcriptome analyses have produced valuable gene expression profiles of hESCs and indicated changes in transcription that occur during differentiation. However, proteins are the actual effectors of these events and changes in their levels do not always match changes in their corresponding mRNA. Furthermore, information on posttranslational modifications that influence the activity of pivotal proteins is still largely missing. Over the years, mass spectrometry has experienced major breakthroughs in high-throughput identification of proteins and posttranslational modifications in cells under different conditions. Mass spectrometry-based proteomic techniques are being applied with increasing frequency to analyze hESCs, as well as media conditioned by feeder cells, and have generated proteome profiles that not only support, but also complement, existing microarray data. In this review, the various proteomic studies on hESCs and feeder cells are discussed. In a meta-analysis, comparison of published data sets distinguished 32 intracellular proteins and 16 plasma membrane proteins that are present in multiple hESC lines but not in differentiated cells, which were therefore likely to include proteins important for hESCs. In addition, 13 and 24 proteins, respectively, were commonly found in different feeder cell lines of mouse and human origin, some of which may be extracellular signaling molecules that play a key role in the undifferentiated propagation of hESCs. These findings underscore the power of mass spectrometry-based techniques to identify novel proteins associated with hESCs by studying these cells in an unbiased, discovery-oriented manner on a proteome-wide scale.