Background: The function of the thymic microenvironment is to promote thymocyte maturation, in part via regulation of thymocyte proliferation and cell death. Defects in fetal thymic epithelial cell (TEC) development and function, and therefore in the formation of a functional microenvironment, can be caused either directly by TEC differentiation defects or indirectly by defective thymocyte maturation. In this paper we studied fetal thymocyte proliferation during the early transition from the CD3-4-8- (triple negative, TN) to CD4+8+ (double positive, DP) stages. We compared wild type mice with Rag1-/- mice and with Hoxa3+/-Pax1-/- compound mutant mice, which have blocks at different stages of thymocyte development. Results: Wild type fetal and adult thymus showed stage-specific differences in the proliferation profiles of developing thymocytes, with fetal stages showing generally higher levels of proliferation. The proliferation profile of fetal thymocytes from Rag1-/- mutants also had stage-specific increases in proliferation compared to wild type fetal thymocytes, in contrast to the lower proliferation previously reported for thymocytes from adult Rag1-/- mutants. We have previously shown that Hoxa3+/-Pax1-/- mice have abnormal fetal TEC development, resulting in increased apoptosis at the TN to DP transition and decreased DP cell numbers. Fetal thymocytes from Hoxa3+/-Pax1-/- compound mutants had increased proliferation, but fewer proliferating cells, at the DP stage. We also observed a decrease in the level of the cytokines IL-7 and SCF produced by Hoxa3+/-Pax1-/- TECs. Conclusion: Our results indicate complex and stage-specific effects of abnormal TEC development on thymocyte proliferation.