CD4⁺ CD8⁺ human thymocytes are functional intermediates between CD3⁻CD4⁺ CD8⁻ progenitors and CD4⁺ CD8β⁺ β-selected pre-T cells. (A) CD3⁻ CD4⁺ CD8⁻thymocytes, isolated as described in Materials and Methods, were cultured in a hybrid hu/mo FTOC and analyzed after 4 and 11 days for the coexpression of CD4, CD8, and CD8β. (B) Intracytoplasmic TCRβ (TCRβic) expression (shaded areas) was analyzed by three-color flow cytometry in the cellular progeny recovered at day 4 of culture. By day 11, analysis was performed after electronic gating on the CD8⁺β⁻ and the CD8⁺β⁺ progeny. Background fluorescence was determined with isotype-matched irrelevant MoAb. A representative experiment out of five is shown.