Severe combined immunodeficient mice transplanted with human organs (SCID-hu mice), provide a unique in vivo model for studying human intrathymic T cell selection and development of tolerance. In vivo administration of staphylococcal enterotoxin B (SEB) to SCID-hu mice causes intrathymic clonal deletion of SEB-specific V beta+ T cells that occurs already at the immature CD4+8+ double positive stage. The expression of activation markers such as CD25, CD71, and HLA-DR was specifically increased on V beta+ T cells responding to SEB. The remaining SEB-specific human T cells that had not been deleted in vivo failed to proliferate when rechallenged with SEB in vitro. These SEB-specific T cells that were rendered anergic in vivo had a unique cytokine production profile. They failed to produce IL-2, which correlated with the lack of proliferation of these cells. In addition, they failed to produce TNF-alpha. However, the anergized T cells synthesized considerable amounts of IFN-gamma, granulocyte-macrophage CSF and IL-10 after SEB stimulation. This clonal anergy can be completely reversed in vitro by stimulating the SEB-specific cells in the presence of exogenous IL-2 or by triggering of the CD28/CTLA-4 activation pathway. Under these stimulation conditions, anergic T cells produced levels of IL-2 and TNF-alpha that were comparable to their non-anergized counterparts, whereas the levels of granulocyte-macrophage CSF, IL-10 and IFN-gamma production were even higher. Collectively, these data demonstrate that in vivo administration of SEB to SCID-hu mice leads to activation, deletion, and anergy of SEB-specific human thymocytes and that the production of IL-2 and TNF-alpha is selectively switched off in these anergic T cells.