Recent results indicate that two signals are required for activation of mature T cells. The first is delivered through the TCR, and the second is delivered through receptors that bind various ligands expressed on APC. For example, it has been shown that B7/BB1, which is expressed on many APC, can costimulate T cell activation by binding to CD28 or CTLA4, which are expressed on mature T cells. In contrast, little is known of the signals required for negative selection of autoreactive thymocytes. Thus, we have investigated this issue by using an in vitro culture system in which thymocytes from mice that are transgenic for a class II MHC-restricted TCR are cultured with murine fibroblast lines that express class II MHC. Under these conditions, CD4+CD8+ (DP) thymocytes undergo an Ag-dependent programmed cell death, which likely represents the negative selection of autoreactive thymocytes that would occur in an intact thymus. Using this culture system, we first found that both TCR- and APC-dependent stimuli were required in order to induce deletion of DP thymocytes. Anti-TCR antibodies alone did not cause deletion of DP cells, but merely induced a decrease in their expression of CD4 and CD8 to produce a DPdull phenotype. Addition of APC was then required for deletion of these DPdull cells. One obvious candidate for the costimulatory signal expressed by these APC was B7. Three different experimental approaches indicated, however, that B7 was not the APC-dependent signal required for deletion of DP thymocytes. Thus, these results suggest that negative selection of autoreactive thymocytes is a two-step process in which stimulation of the TCR causes downregulation of CD4 and CD8 on DP thymocytes, and then an unknown ligand expressed on APC stimulates a receptor on DP thymocytes to induce their deletion.