To date, technical limitations have precluded the robust quantitative proteomic analysis of rare cell types. We describe a highly sensitive mass spectrometry-based proteomic workflow for the analysis of human hematopoietic stem cells and three progenitor cell types. More than 5,000 protein groups could be consistently quantified from 25,000 sorted hematopoietic stem and progenitor cells. The data reproducibly identified characteristic patterns of differentially expressed proteins in the tested populations that indicated biochemical differences not apparent by transcriptomic analyses on equivalent samples.