T-lineage differentiation can be elicited from various myeloid–lineage-committed or DC-committed progenitors. (A) BM-derived and thymic progenitors were sorted from CX3CR1^GFP/+ reporter mice. Purified precursors were cultured on OP9-DL1 stromal cells in the presence of Flt-3L, SCF, and IL-7, and analyzed at days 4, 6, and 8 to assess their myeloid- and T-lineage potential. DCs were considered as CD11c⁺, while macrophages were CD11b⁺CD11c⁻. T-cell–committed precursors were negative for myeloid markers (CD11c, CD11b, and CX3CR1) and further subdivided into 3 DN populations based on the surface expression of CD25 and CD44. DN1 were CD44⁺CD25⁻, DN2 CD44⁺CD25⁺, and DN3 CD44⁻CD25⁺. (B) Quantification of data shown in (A) at day 4 (upper panel) and day 6 (bottom panel) of culture. Numbers above the columns indicate fold expansion of precursors. (C) Quantification of T-lineage–committed DN populations shown in (A) at day 4 (upper panel) and day 6 (bottom panel) of culture. (D) The potential of CX3CR1⁺ and CX3CR1⁻ ETPs to develop toward the T lineage was assessed by limiting dilution assay on OP9-DL1 stromal cells in the presence of Flt-3L, SCF, and IL-7. (A-D) Data are representative of 2 independent experiments.