The lineage restriction of prospectively isolated hematopoietic progenitors has been traditionally assessed by bulk in vitro culture and transplantation of large number of cells in vivo. These methods, however, cannot distinguish between homogenous multipotent or heterogeneous lineage-restricted populations. Using clonal assays of 1 or 5 cells in vitro, single-cell quantitative gene expression analyses, and transplantation of mice with low numbers of cells, we show that a common myeloid progenitor (CMP) is Sca-1^lolin⁻c-Kit⁺CD27⁺Flk-2⁻ (SL-CMP; Sca-1^lo CMP) and a granulocyte/macrophage progenitor (GMP) is Sca-1^lolin⁻c-Kit⁺CD27⁺Flk-2⁺CD150^−/lo (SL-GMP; Sca-1^lo GMP). We found that mast cell progenitor potential is present in the SL-CMP fraction, but not in the more differentiated SL-GMP population, and is more closely related to megakaryocyte/erythrocyte specification. Our data provide criteria for the prospective isolation of SL-CMP and SL-GMP and support the conclusion that mast cells are specified during hematopoiesis earlier than and independently from granulocytes.