Among the several mechanisms known to be involved in the establishment and maintenance of immunological tolerance, the activity of CD4+CD25+ regulatory T lymphocytes has recently incited most interest because of its critical role in inhibition of autoimmunity and anti-tumor immunity. Surprisingly, very little is known about potential genetic modulation of intrathymic regulatory T lymphocyte-development. We show that distinct proportions of CD4+CD25+FoxP3+ regulatory T cells are found in thymi of common laboratory mouse strains. We demonstrate that distinct levels of phenotypically identical regulatory T cells develop with similar kinetics in the studied mice. Our experimental data on congenic mouse-strains indicate that differences are not caused by the distinct MHC haplotypes of the inbred mouse strains. Moreover, the responsible loci act in a thymocyte intrinsic manner, confirming the latter conclusion. We have not found any correlation between thymic and peripheral levels of regulatory T cells, consistent with known homeostatic expansion and/or retraction of the peripheral regulatory T cell pool. Our data indicate that polymorphic genes modulate differentiation of regulatory T cells. Identification of responsible genes may reveal novel clinical targets and still elusive regulatory T cell-specific markers. Importantly, these genes may also modulate susceptibility to autoimmune-disease.