Activation of conventional DC1 and DC2 requires distinct signals. (A) scRNA-seq of CD11c⁺ and CD11b⁺ FACS-sorted cells from the thymus of 7-wk-old C57BL/6 mice. Cells were bioinformatically filtered to include only clusters expressing Flt3. UMAP plots show the analysis of 6,928 transcriptome events, identifying 11 clusters. (B) Feature plot showing the normalized expression of Flt3 in clusters identified in Fig. 3 A. (C) Representative flow cytometry plots identifying CCR7⁺ and XCR1⁺ pDCs in the thymus. The graph shows frequency of CCR7⁺ and XCR1⁺ cells within the thymic SiglecH⁺ population; n = 4 mice. (D) Representative flow cytometry plots showing the normalized expression of CD80, CD86, CD40, PD-L1, and CD63 in thymic DC populations defined in Fig. 3 B. (E) Feature plots showing the normalized expression of Xcr1 and Sirpa in clusters identified in Fig. 3 A. (F) Representative flow cytometry plots showing expression of XCR1 in eYFP⁺ cells from Xcr1^iCreRosa26^eYFP (Xcr1^eYFP) mice. The graph shows frequency of CCR7⁺ cells in cell populations defined by flow cytometry; n = 4 mice, from two independent experiments. (G) Representative flow cytometry plots comparing the thymic DCs (gated as in Fig. 3 C) in Batf3^−/− and Zeb2^Δ1+2+3 mice. (H) Numbers of thymic DCs (gated as in Fig. 3 C) in Ifnar1^−/− and Ifnlr1^−/− mice, shown as KO/WT ratio of cell numbers; n = 8–11 mice from at least three independent experiments. Data are shown as mean ± SD. Statistical analysis was performed by a one-sample t test and Wilcoxon test with a theoretical mean of 1, *P ≤ 0.05, ***P ≤ 0.001, and n.s., not significant.