We have previously reported that human granulocyte-macrophage colony-stimulating factor (hGM-CSF) causes a stage-specific inhibition of T-cell receptor (TCR) alphabeta cell development in the thymus of transgenic mice constitutively expressing the hGM-CSF receptor. Since it has been reported that the addition of interleukin-7 (IL-7) to fetal thymic organ culture (FTOC) has similar effects, we compared the effects of IL-7 and hGM-CSF on TCR(alphabeta) cell development in hGM-CSF receptor transgenic mice. We reconstituted fetal lobes with sorted pre-T, or post pre-T CD4(-)CD8(-) precursor cells. The addition of either IL-7 or hGM-CSF to these cultures suppressed further differentiation of pre-T cells but not post pre-T cells. At the same time, the cell number was increased, suggesting that pre-T-cell proliferation is stimulated by these cytokines. Furthermore, the differentiation of recombination-activating gene-1 (RAG-1)-deficient pre-T cells in response to anti-CD3 antibody stimulation was suppressed by either IL-7 or hGM-CSF, suggesting that these cytokines inhibit the pre-T-cell receptor (pre-TCR) signal. This inhibition is unexpected because the pre-TCR signal and the IL-7 signal have previously been considered to be co-operative. Recent analysis of the downstream events of IL-7 receptor and GM-CSF receptor revealed that they share common signal transduction molecules. Our results show that IL-7 is able to promote pre-T cell proliferation and to suppress differentiation induced by the pre-TCR signal. GM-CSF can mimic these biological activities of IL-7 when the pre-T cells express GM-CSF receptors. Our data suggest that both timing and level of activation of the IL-7 signalling pathway must be precisely regulated to facilitate the differentiation of thymocytes.