Human thymocyte differentiation was examined by injecting fetal thymic progenitor populations into human thymic xenografts in SCID-hu mice. Thymic progenitors were fluorescently labeled with the lipophilic dye PKH2. The phenotypes of their progeny could be identified by flow cytometric analysis of cells with a very high fluorescent PKH2 signal. Intrathymic injection of purified triple negative (TN) CD3-4-8- thymocytes resulted in the sequential appearance of CD3-4+8-, CD3-4+8+, and CD3+4+8+ cells, with the subsequent appearance of small numbers of phenotypically mature CD3+4+8- and CD3+4-8+ cells over a 4-d period. Sorted CD3-4+8- thymocytes injected intrathymically rapidly differentiated to CD4+8+ cells. CD4+8+ fetal thymocytes in cell cycle differentiated into phenotypically mature CD3+4+8- and CD3+4-8+ populations, whereas nondividing CD4+8+ cells failed to differentiate after intrathymic transfer. The number of cell divisions that occurred between the injection of TN thymocytes and their progeny at different time points was estimated based on the decrease in the intensity of the PKH2 label. The average length of the cell cycle for the TN population was calculated to be 24 h. The SCID-hu model thus provides a useful tool for studying the kinetics of cell division and differentiation of human thymocytes in vivo.