Mature SP T cells migrate away from the thymic stroma/CD34⁺CD2^– cell coculture in which they are generated. Purified human bone marrow–derived CD34⁺CD2^– hematopoietic progenitor cells were plated onto confluent murine thymic stroma established on the three-dimensional matrix Cellfoam to form a thymic organoid. A sample of cells was harvested from inside and outside the thymic organoid under microscopic guidance at day 4 (a) and day 14 (b) and stained with anti-CD4 (FITC-labeled) and anti-CD8 (PERCP- or PE-labeled). The thymic organoid was also cultured in the presence of PTX (10 ng/ml) between day 7 and day 14, and cells were harvested and analyzed at day 14 (c). At day 14 of the coculture, cells began to appear at the edge of cultures away from the thymic stroma–coated Cellfoam. At day 14 the majority of cells from the periphery of the coculture were CD4⁺CD8^– or CD4^–CD8⁺, whereas the majority of cells within the Cellfoam were CD4⁺CD8⁺ (b). At day 14, in the culture containing the thymic organoid exposed to PTX, the majority of cells were detected in the grid and were either CD4⁺CD8⁺ and CD4⁺ CD8^– or CD4^–CD8^– thymocytes. A very small proportion of CD4⁺CD8^– and CD4^–CD8^– thymocytes was detected outside of the thymic organoid in these PTX-treated cultures (c). The percentage of cells falling into each immunophenotypic category is shown in the top right corner of each quadrant. *P < 0.05, **P < 0.01, Student t test.