Abstract TOX (Thymocyte-selection associated HMG-box) is a DNA-binding protein identified in our laboratory that is required for development of the CD4 T cell lineage, including canonical CD4 T cells, NKT cells and T regulatory (Treg) cells. In addition, NK cell and lymphoid tissue inducer cell development requires TOX, the latter resulting in failure of lymph node development in TOX-deficient mice. Here we turn our attention to investigation of TOX in mature CD4 T cell subsets, facilitated by a knock-in Tox reporter (tdTomato) strain of mouse. The reporter faithfully mirrors TOX expression in the thymus and NK cells. We find that TOX (Tomato) is also upregulated in natural Treg and at highest levels in T follicular helper (Tfh) cells. Interestingly, both T cell subsets show heterogeneity in reporter expression, with cell populations of high, intermediate and low expression evident. In Treg, varied expression of TOX is independent of FOXP3 expression, as demonstrated by analysis of FOXP3 and TOX dual reporter mice. Among Tfh cells (CXCR5hiPD1hi), Toxhi cells are Bcl6hiCCR7-CD62Llo and likely represent mature follicular effector cells. To study the functional role of TOX, we expressed Cre in CD4 T cells from OTII Tox conditional knockout mice, and analyzed the development of phenotypically-defined Tfh in response to antigen upon adoptive transfer. Loss of TOX prevented Tfh cell development in this system, indicating a critical role for TOX in this peripheral T cell subset.