The deletion of the POZ domain of Miz-1 disrupts T-cell development at the transition from DN to DP and at the ETP/DN1 stage. Flow cytometric (FACS) analysis (A,D) and total cell numbers (B,C,E-G) of thymic populations of wild-type (WT) and Miz-1^ΔPOZ mice. (A) CD4 and CD8 surface staining (top panel) and lineage-negative (Lin⁻) cells (bottom panel), described in supplemental Methods are analyzed for the surface expression of CD44 and CD25 to assess DN1 (Lin⁻CD44⁺CD25⁻), DN2 (Lin⁻CD44⁺CD25⁺), DN3 (Lin⁻CD44⁻CD25⁺), and DN4 (Lin⁻CD44⁻CD25⁻). Numbers in quadrants indicate the percentage of cells. (B-C) Absolute numbers of thymocytes, TCR-αβ⁺, TCR-γδ⁺, and DN1 to DN4 cells are calculated relative to the live cells gated according to the FSC/SSC profile and expressed as absolute cell count. (D) FACS analysis of ETP (Lin⁻ no CD4, CD25⁻ CD44⁺CD117⁺), DN1a-e (Lin⁻ no CD4, CD25⁻, CD44⁺, and CD24/CD117^medium/high) and DN2a-b (Lin⁻ no CD4, CD44⁺CD117⁺CD25^medium/high) populations. (E-G) Percentages of positive cells in panel D are calculated relative to the total live cells and expressed as absolute cell count. Average counts of at least 8 mice and error bars representing the SD are shown. **P ≤ .01. ***P ≤ .001.