Thymocytes display varying responses to cytokines depending on their stage of differentiation. Whether these responses are due to stage-specific cytokine receptor expression or to downstream signaling mechanisms is unknown. We examined the relationship between receptor expression and thymocyte proliferation or differentiation by using thymocytes from transgenic mice that constitutively expressed the human granulocyte-macrophage colony stimulating factor (hGM-CSF) receptor. Transgenic CD4^-CD8^-, CD4⁺CD8^-, and CD4^-CD8⁺ thymocyte populations expressing the hGM-CSF receptor proliferated when cultured with hGM-CSF, whereas CD4⁺CD8⁺ cells failed to proliferate despite expressing this receptor. We next examined the effect of hGM-CSF receptor signaling on thymocyte differentiation in fetal thymic organ culture supporting a full program of T cell development in vitro. Addition of hGM-CSF to the transgenic fetal thymic organ culture resulted in failure of CD4^-CD8^- cells to differentiate into CD4⁺CD8⁺ cells. To investigate this maturational inhibition more closely, we repopulated wild-type fetal lobes with sorted pro-T, pre-T or post pre-T precursor cells from hGM-CSF receptor transgenic mice. In these cultures hGM-CSF blocked both pro-T and pre-T cell differentiation, whereas the more mature post pre-T cells differentiated normally. These results suggest that hGM-CSF receptor signaling during thymocyte differentiation causes stage-specific inhibition of precursor cell maturation. In addition, repopulation studies of transgenic fetal lobes with sorted wild-type thymocyte precursors indicated that hGM-CSF inhibited proliferation and differentiation of the wild-type precursors, suggesting a secondary effect via transgenic stromal cells. (J Allergy Clin Immunol 1997;100:S87-96.)