Hoxb4 is broadly expressed in embryonic HSCs from the E14.5 fetal liver. (A) FL cells were gated using Lin⁻ and Sca1⁺ markers and sorted on the basis of YFP expression. Recovered cells were tested in long-term reconstitution experiments (200 or 1000 cells injected). (B) Donor contribution to the peripheral blood is displayed from 2 independent sorting experiments. (C) Assessment of Hoxb4/YFP expression in the donor-derived CD45.2⁺ LSK fraction from recipient CD45.1 mice reconstituted with sorted FL HSCs 16 weeks after transplantation. (D) Irradiated recipients were injected with one E14.5 FL to obtain > 90% peripheral blood reconstitution. BM from reconstituted mice was sorted as in Figure 2C into Lin⁻Sca1⁺Hoxb4/YFP⁺ and Lin⁻Sca1⁺ Hoxb4/YFP⁻ populations and injected into irradiated recipients. Contribution to the peripheral blood was determined by flow cytometry. HSCs were only observed in the Hoxb4/YFP-expressing cell fraction. Red histo-grams represent control BM LSK cells, and blue histograms show LSK CD45.2 cells in BM from Hoxb4/YFP-reconstituted CD45.1 recipients. Result from one experiment.