Hematopoietic stem cells differentiate in the thymus to T cells along precisely defined intermediates. This process is thymic epithelium dependent and involves cytokines and cell-cell interactions between thymic stroma and T-cell precursors. Here we report that highly purified human CD34++ fetal liver stem cells differentiate to mature T cells, when seeded into isolated fetal thymic lobes of severe combined immunodeficient mice, and subsequently cultured in vitro. The human stem cells differentiate sequentially into CD4+CD8-CD3-, CD4+CD8+CD3-, CD4+CD8+CD3+, and finally, CD4+CD8-CD3+4 and CD4-CD8+CD3++ cells. Phenotypic analysis for additional maturation markers showed that these CD4 and CD8 single-positive thymocytes are fully maturate cells. By immunochemistry, human HLA-DR+ cells with a dendritic morphology could be detected. This novel chimeric human-mouse fetal thymus organ culture offers a tool to study human T-cell ontogeny in vitro and is a rapid and reliable test method for T-cell precursor activity of cultured or transfected human stem cells.