Immunoregulatory CD4− CD8− (double-negative; DN) T cells exhibit a unique antigen-specific mode of suppression, yet the ontogeny of DN T cells remains enigmatic. We have recently shown that 3A9 T-cell receptor (TCR) transgenic mice bear a high proportion of immunoregulatory 3A9 DN T cells, facilitating their study. The 3A9 TCR is positively selected on the H2k MHC haplotype, is negatively selected in mice bearing the cognate antigen, namely hen egg lysozyme, and there is absence of positive selection on the H2b MHC haplotype. Herein, we take advantage of this well-defined 3A9 TCR transgenic model to assess the thymic differentiation of DN T cells and its impact on determining the proportion of these cells in secondary lymphoid organs. We find that the proportion of DN T cells in the thymus is not dictated by the nature of the MHC-selecting haplotype. By defining DN T-cell differentiation in 3A9 TCR transgenic CD47-deficient mice as well as in mice bearing the NOD.H2k genetic background, we further demonstrate that the proportion of 3A9 DN T cells in the spleen is independent of the MHC selecting haplotype. Together, our findings suggest that immunoregulatory DN T cells are subject to rules distinct from those imposed upon CD4 T cells.