We have previously identified a nuclear factor member of the HMG‐box superfamily, designated TOX (thymocyte selection‐associated HMG box protein), which is transiently expressed in the thymus during both positive selection and β‐selection. We now report that mice deficient in TOX (Tox^−/−) have severe reductions in all CD4 lineage T cells, including conventional T, regulatory T and NK‐T cells, due to a block in the CD4^loCD8^lo to CD4⁺CD8^lo thymocyte transition of positive selection. We did not detect a TCR signaling defect in Tox^−/− thymocytes and loss of the CD4 T cell lineage is not due to increased cell death, as expression of the anti‐apoptotic protein BCL‐2 does not rescue the development of CD4 T cells. The loss of the CD4 lineage, however, is associated with failure to upregulate Zbtb7b, but not GATA3, during positive selection. In contrast, functional CD8 T cells can develop in Tox^−/− mice. The block in development of CD4 but not CD8 T cells in Tox^−/− mice is also observed in TCR transgenic animals. Moreover, the failure of class II MHC restricted T cells to be misdirected into the CD8 lineage in these mutant mice has implications for the mechanism of lineage commitment in the thymus. These results demonstrate differential requirements for the CD4 and CD8 lineages, where CD4‐destined cells require induction of TOX and passage through a CD4⁺CD8^lo stage for their development, while CD8‐destined cells can develop directly from CD4^loCD8^lo thymocytes. We are currently working to identify the changes in gene expression mediated by TOX.