Ectopic expression of insulin in thymus has been suggested to be involved in tolerance induction against pancreatic beta cells and in type 1 diabetes (T1D) pathogenesis. However, it is not known whether thymic insulin expression would also influence thymocyte maturation and differentiation. To address these questions, we have used mice that are insulin deficient. Early fetal thymi were cultured in fetal thymic organ cultures (FTOCs) and the development of thymocytes was studied by flow cytometry. The results revealed no significant difference in thymocyte maturation in the absence of thymic insulin. Taken together, these data do not support a role for thymic insulin in thymocyte differentiation and growth.