GATA-3, PLZF, and Bcl11b are required after the EILP stage. (A–D) Flow cytometric analysis of bone marrow cells from Vav1-iCre Gata3^f/f mice and Vav1-iCre Gata3^+/+ littermate controls. (A) Profiles of EILPs after gating on Lin⁻ Kit⁺ CD122^low cells. (B) α4β7 and IL-7rα expression of GATA-3–sufficient (black) and –deficient (red) EILPs as gated in A. Kit⁺ Lin⁻ cells were used as a negative control for IL-7rα expression (gray). (C) Quantification of ALP, EILP, and ILCp numbers normalized using ALPs (Fig. S5). (D) Profiles of ILCps as defined as PLZF⁺ α4β7⁺ after gating on Lin⁻ Kit⁺ Flt3⁻ cells. (E and G) Flow cytometric analysis of bone marrow cells from Zbtb16^−/− mice and Zbtb16^+/+ littermate controls. (E) Quantification of ALP, EILP, ILCp, and ILC2p numbers normalized using ALPs (Fig. S5). (F) Profiles of ILC2p after gating on Lin⁻ Kit⁻ CD122^low cells. (A, D, and F) Numbers indicate the percentage of cells in each gate. (G) α4β7 and IL-7rα expression for PLZF-sufficient (black) and -deficient (red) EILPs or ILC2ps gated in F. (H) ILC development analyzed by flow cytometry in the bone marrow of long-term competitive chimeras reconstituted for 10–12 wk with Bcl11b^+/+ or Bcl11b^−/− fetal liver LSK cells (CD45.2) mixed with equal numbers of WT bone marrow LSK cells (CD45.1). ILC2ps are gated as Lin⁻ Kit⁻ Sca1^high Flt3⁻ IL-7Rα⁺ cells. Donor chimerism of the indicated population was normalized to bone marrow ALP chimerism for individual mice. (C, E, and H) Data are representative of at least two independent experiments and are presented as mean ± SEM for n = 3 mice per group. A two-tailed unpaired Student’s t test was performed to determine significance. *, P < 0.05; **, P < 0.01. (I) Breakdown of progenitors during early ILC development. Progenitor–successor relationships between cell types are indicated by arrows. The requirements for key transcription factors at developmental transitions are specified.