Identification of ETPs expressing CX3CR1. (A) Thymocytes from CX3CR1^GFP/+ reporter mice were depleted of CD4 and CD8 positive cells and subsequently stained with lineage antibodies (CD19, TCR-β, NK1.1, CD11b, CD11c, and Gr-1), as well as CD44, CD25, and CD117. Representative FACS density plots show ETPs (lin⁻CD44⁺CD25⁻CD117^hi) negative and positive for CX3CR1. (B) ETPs prepared as in (A) positive (red, R1) or negative (black, R2) for CX3CR1 and were tested for expression of markers associated with myeloid precursors: CD135, CD115, CD11c, and CD11b. FMO controls were used to visualize background fluorescence. (C) Thymocytes were prepared and stained as in (A) and antibodies against CD24 to discriminate DN1 subsets according to the DN1a-e scheme. (D) Levels of CD24 on DN1 cells. Numbers inside contour plots indicate median fluorescent intensities of the respective surface antigens. Data are representative of 6 (A) and 2 (B-D) independent experiments. FMO, fluorescence-minus-one.