The fields of mass spectrometry (MS) and stem cell biology have expanded greatly in the past twenty years. Taken alone, these fields occupy entirely different branches of science; however, the points where they overlap provide valuable insight, both in the biological and technical arenas. From a biological perspective, MS-based proteomics offers the capacity to follow post-transcriptional regulation and signaling that are (1) fundamental to pluripotency and differentiation, (2) largely beyond the reach of genomic technologies, and (3) otherwise difficult or impossible to examine on a large scale. At the same time, addressing questions fundamental to stem cell biology has compelled proteomic researchers to pursue more sensitive and creative ways to probe the proteome, both in a targeted and high-throughput manner. Here, we highlight experiments that straddle proteomics and stem cell biology, with an emphasis on studies that apply mass spectrometry to dissect pluripotency and differentiation.