Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method, we analyzed over 1,500 single cells throughout the mouse hematopoietic system and illustrate its utility for revealing important biological insights. The comprehensive single cell data set permits mapping of the mouse hematopoietic stem cell differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems. Display omitted •Robust methodology for single-cell analysis of the cell surface repertoire•Comprehensive single-cell analysis for the mouse hematopoietic system•Lineage progression analysis and network construction using single-cell data•Characterization of the unique cellular hierarchy in a mouse AML model Single-cell analysis of the cell surface repertoire enables mapping of cellular heterogeneity and differentiation hierarchy, applied here to the hematopoietic system but with broad applicability.