(A) Nfil3−/− mice show reduced frequencies (left panel) and numbers (right panel) of major ILC types, including conventional NK (cNK), non-NK ILC1, ILC2 and ILC3. Lymphocytes were isolated from the small intestinal lamina propria and the liver and were stained as described in Materials and methods. Gating strategies are depicted in Figure 1—figure supplement 1. cNK cells were identified as CD45+ Lin(CD3ε, CD19, CD5, TCRβ, TCRγδ)- NK1.1+ T-BET+ EOMES+; non-NK ILC1 as CD45+ Lin(CD3ε, CD19, CD5, TCRβ, TCRγδ)- NK1.1+ T-BET+ EOMES−; ILC2 as CD45+ Lin(CD3ε, CD19)− GATA3+ Sca1+ KLRG1+; and ILC3 as CD45+ Lin(CD3ε, CD19)- RORγt+ CD127+. The NK receptor-expressing subtype of ILC3 (also known as NK22 cells) was identified by additional staining for NKp46. (B) NFIL3 regulates ILC development in a bone-marrow progenitor intrinsic manner. Equal numbers of wild-type (CD90.2+ CD45.1+) and Nfil3−/− (CD90.2+ CD45.2+) LSK cells were co-transplanted into lethally irradiated CD90.1+ mice. Liver CD90+ NK and non-NK ILC1 and intestinal ILC2 and ILC3 were analyzed 4-6 weeks later. The ratios of ILCs derived from wild-type (CD45.1+) and Nfil3−/− (CD45.2+) donor cells were calculated and plotted. Significant variation from 1.0 is indicated by *. sm. int., small intestine. (C) Nfil3 regulates ILC development in a CLP-intrinsic manner. Equal numbers of wild-type (CD45.1+) and Nfil3−/− (CD45.2+) CLPs were co-transplanted into sublethally irradiated alymphoid Rag2−/−;Il2rg−/− mice. ILCs were analyzed 4–6 weeks later as for the LSK experiment. Groups were compared by two-tailed student's t-test (A), one-sample t-test (B, LSK) or Wilcoxon signed rank test (B, CLP). Means ± SEM are shown. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.