Gli3 is downstream of pre-TCR signaling for thymocyte differentiation. (A) Two-day FTOCs of E15.5 Rag1^–/–Gli3^–/– and Rag1^–/–Gli3^+/+ thymi with anti-CD3 antibody were performed and stained for CD4 and CD8 expression. The percentage of DP thymocytes was reduced in Rag1^–/–Gli3^–/– relative to the Rag1^–/–Gli3^+/+ control. Percentages of thymocytes in each quadrant are shown. (B) Mean percentages of DN, CD8 ISP, DP, and SP CD4 cells induced in the Rag1^–/–Gli3^+/+, Rag1^–/–Gli3^+/–, and Rag1^–/–Gli3^–/– FTOCs treated with anti-CD3 are shown. The differences observed between Rag1^–/–Gli3^+/+ and Rag1^–/–Gli3^–/– are statistically significant according to Student t test P values (< .05). Error bars represent the standard error of the mean.