We previously reported that IL-7 maintains the viability and differentiation potential of CD25 (IL-2R p55) positive CD3-CD4-CD8- thymic pre-T cells in vitro. This culture system is suitable for studying signals that regulate differentiation of T cell precursors in the thymus. In this study, we screened cytokines for their capacity to induce CD4 or CD8 in murine thymic pre-T cells cultured with IL-7. Of 15 cytokines tested, only transforming growth factor (TGF-beta) and TNF-alpha induced CD8 (Lyt-2), while no cytokine was able to induce CD4 on CD25+CD3-CD4-CD8- thymocytes. The combination of TGF-beta and TNF-alpha was synergistic, and the majority of cells recovered after 2 to 3 days in culture expressed CD8 (but not CD3 or CD4). A similar effect of TGF-beta and TNF-alpha was observed using day-15 fetal thymocytes, CD3+CD4-CD8- or CD3+CD4+CD8- adult thymocytes, although the combination of these cytokines resulted in an additive rather than a synergistic effect in these subsets. In contrast, neither TGF-beta nor TNF-alpha induced CD8 expression on splenic CD4+CD8- T cells. These observations suggest a role for these cytokines in the induction of CD8 expression in CD8- thymocyte subsets including CD3-CD4-CD8- thymic pre-T cells.