T-lineage potential is confined specifically in the Flt3⁺CD150^– preGM subset of CMPs. (A) Previously described CMPs can be further subdivided into 3 populations based on additional Flt3 and CD150 expression. (B) Three subsets of CMPs, along with MPPs, were double FACS-sorted, seeded at equal number (300 cells per well), and cultured on OP9 or OP9DL4 for 10 days to assess the DC, myeloid, B, and T-lineage potentials. DC and myeloid lineages were identified based on CD11c and Mac1 expression, respectively, and both lineages could be readily detected from MPPs and Flt3⁺CD150^– preGMs that were cocultured on OP9 or OP9DL4. B-lineage progeny was identified as Mac1^–CD11c^–CD19⁺B220⁺ cells, whereas T-lineage cells were Mac1^–CD11c^–CD25⁺Thy1⁺. Of 4 populations of progenitors tested here, only MPPs could generate B cells and T cells on OP9 and OP9DL4, respectively. No B-lineage precursor activity could be detected in Flt3⁺CD150^– preGMs, although they could give rise to T cells on OP9DL4. Flt3^–CD150^– preGMs appeared myeloid restricted, as they only give rise to Mac1⁺ myeloid cells exclusively but no other lineages on OP9 and OP9DL4. CD150⁺ preMegEs did not give rise to lymphoid or myeloid cells. (C) More than half of Flt3⁺CD150^– preGM progenitors are T/myeloid bi-potent. Single cells of Flt3⁺CD150^– preGMs or MPPs were deposited by FACSAria sorter onto mixed OP9/OP9DL4 stromal cells and cultured in the presence of additional cytokines (see “OP9 cultures and single-cell cultures”) for 10 days before they were analyzed for myeloid (Mac1) or T-lineage (CD25 or Thy1) development. 147 clones of MPPs and 162 clones of Flt3⁺CD150^– preGMs were analyzed with 88% and 85% cloning efficiency, respectively. Derivation of cloning efficiency for the single-cell assay is detailed in “Methods.” Results shown are from at least 3 independent experiments and single-cell culture results are from 2 independent experiments.