Total CMPs harbor in vitro T-lineage potential. (A) Traditional CMPs were identified and sorted by flow cytometry. BM from WT B6 mice was stained for Lin, Sca1, Kit, CD34, and FcγRII/III (CD16/32). CMPs were defined as Lin^–Sca1^–Kit⁺CD34⁺FcγRII/III^low.³  Multipotent LSK cells were sorted as Lin^–Sca1⁺Kit⁺. (B) CMPs or LSK cells (300 cells) were sorted from the BM of NG-BAC mice, a Rag2-reporter strain, and cultured on OP9DL1 for 7 days, before flow cytometric analysis of Mac1/Gr1, CD25, and GFP^Rag2 expression. (C) FACS-sorted total CMPs (300 cells), along with LSK cells, from WT B6 BM were seeded onto OP9DL1 (or OP9DL4) stromal cells. The cocultures were passaged every 5-7 days. Live hematopoietic cells (DAPI^–CD45⁺GFP^– gated) deriving from LSK cells or CMPs on OP9DL1 after 28 days of coculture were assessed for T-lineage development based on CD4, CD8, CD25, CD44, and TCRβ expression. Normal B6 thymocytes were included as reference. Shown are representative results of at least 3 independent experiments. (D) Number of CD4⁺CD8⁺ DP and CD4^–CD8^–CD25⁺ DN3 cells obtained in experiments shown in panel C. *P = .04, **P = .01. Error bars denote SEM. P values were determined by Student t test. (E) CMPs, in addition to LSK cells, GMPs, and MEPs, were seeded on OP9DL4 at increasing numbers per well. LSK cells established a 1/3.2 T-lineage precursor frequency on OP9DL4, while the T-lineage precursor frequency from total CMPs registered at 1/28. No T-lineage precursor frequency could be determined for GMPs. Results shown are from 4 independent experiments.