Gli3 is not involved in survival, proliferation, or lineage commitment of fetal thymocytes. (A) Thymocyte number isolated from Gli3^–/–, Gli3^+/–, and Gli3^+/+ embryos is not significantly different at embryonic days E14.5, E15.5, and E16.5. (B-C,E-F) ▪ indicates Gli3^+/+; ▦, Gli3^+/–; and □, Gli3^–/–. Error bars represent the standard error of the mean. (B) Cell death (as measured by annexin V staining) was not significantly different in any thymocyte population isolated from E16.5 Gli3^+/+, Gli3^+/–, and Gli3^–/– mice. (C) Cell death measured as in panel B of DN3 and DN4 cells isolated from E15.5 Gli3^+/+, Gli3^+/–, and Gli3^–/–. No significant differences were observed. (D) Propidium iodide staining was used to assess cell-cycle status of total thymocytes isolated from E16.5 Gli3^+/+, Gli3^+/–, and Gli3^–/– mice. Percentages of thymocytes at the G₂/S phases of the cell cycle are shown. (E) Cell-cycle status analyzed by staining with DRAQ5 simultaneously with cell-surface markers for a combination of CD4, CD8, CD44, and CD25. No significant differences in cell-cycle status was observed in any thymocyte subset analyzed from E16.5 Gli3^+/+, Gli3^+/–, and Gli3^–/– embryos. (F) Cell-cycle status of E15.5 DN3 and DN4 was analyzed as in panel E. There were no significant differences observed between Gli3^+/+, Gli3^+/–, and Gli3^–/– embryos. (G) E16.5 thymocytes were stained with anti-TCRγδ and anti-NK1.1 antibodies. No significant differences were observed. Percentages of TCRγδ or NK1.1 CD44^–CD25^– DN cells are shown.