The influence of recombinant interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF) on IL-4-induced proliferation of postnatal human thymocytes from eight children with Down syndrome (DS, trisomy 21) and 18 control children was evaluated. DS thymuses were studied because they are characterized by cortical depletion and abnormal thymocyte differentiation. IL-4, without mitogen, induced a dose-dependent proliferation of both DS and control thymocytes. The proliferation was comparable to that induced by IL-2 and far greater than the proliferation mediated by IL-1β in the absence of mitogen. The level of IL-4 responsiveness correlated with the proportion of cells expressing the γ,δ chains of the T cell receptor. Furthermore, thymocyte preparations greatly enriched for T cell receptorγ,δ-bearing cells were found to vigorously proliferate when treated with IL-4. Both IFN-γ and TNF inhibited IL-4-driven proliferation in a dose-dependent manner, but DS thymocytes were found to be significantly more sensitive to inhibition by both cytokines. Our studies suggest an important role for IL-4 in the proliferation of T cell receptorγ,δ+thymocytes and demonstrate regulatory functions for IFN-γ and TNF in human thymocyte proliferation. The increased sensitivity of DS thymocytes to IFN-γ and TNF may explain anatomical abnormalities in DS thymuses and suggests the involvement of genes encoded on human chromosome 21 in the responses to both IFN-γ and TNF. (Pediatr Res 32: 269–276, 1992)