Thymopoiesis-initiating cells develop exclusively from HSC-derived progenitors. (A) Experimental design of lineage-tracing analysis of Csf1r-expressing cells after OH tamoxifen (OH-TAM) administration at E8.5, E9.5, or E10.5. Arrows indicate pulse and analysis time points. (B) Flow cytometry analysis of E12.5 early thymic progenitors (ETPs). E12.5 thymic lobes of embryos pulsed at indicated time points were analyzed. Left plots show the gating strategy to identify ETP. Right representative histograms show the frequency of Tomato⁺ or YFP⁺ cells in ETP. For each experiment, 1 representative analysis is shown. (C) Frequencies of YFP- or Tomato-labeled hematopoietic progenitors and LTi’s in E12.5 FL, fetal blood (FB), and thymi (left plots) and of LSK, B1 B cells, B cells, and Vγ5⁺ T cells in adult tissues (right plots) of animals pulsed at E8.5, E9.5, or E10.5. Microglia served as controls for labeling efficiency. All data are pooled from minimally 2 independent experiments for embryos analyzed at E12.5 (pulsed at E8.5, n = 8; E9.5, n = 10; E10.5, n = 10); for adult tissues, animals were analyzed between 10 and 12 weeks of age (pulsed at E8.5, n = 6; E9.5, n = 6; E10.5, n = 2). Data are depicted as mean plus or minus SEM except for adult animals pulsed at E10.5 (mean). BM, bone marrow; FT, fetal thymus; PerC, peritoneal cavity.