Defective thymus reconstitution by DKO lin⁻ BM cells is not the result of impaired intrathymic T-cell differentiation. (A) In vitro T-lineage differentiation of wt and CCR7^−/−CCR9^−/− (DKO) lin⁻ BM cells. Lin⁻ BM cells were cocultured with OP9-DL4 stromal cells (1 ng/mL interleukin-7, 5 ng/mL Fms-like tyrosine kinase 3 ligand, and 10 ng/mL stem cell factor), and T-lineage commitment was assessed flow cytometrically by staining for CD25 and CD90.2 after 7 days. DP cells were considered T-lineage committed. Data represent mean ± SEM (n = 12). (B) Lin⁻ BM competitor cells (CD45.1/CD45.2) were mixed with an equal amount of lin⁻ BM cells (CD45.2) from wt or various mutant mice. Mixtures of competitor and test cells were transferred intravenously into B6 CD45.1 mice of approximately 35 days of age, and the phenotype of donor-derived thymocytes (CD45.2) was analyzed by staining for CD4 and CD8 21 days after transfer. (C) A total of 10⁵ lin⁻ BM cells (CD45.2) from wt or various mutant mice were transferred intrathymically into B6 CD45.1 mice, and frequencies of donor-derived thymocytes were assessed 21 days after transfer by staining for CD45.1, CD45.2, CD4, and CD8. Results are representative of 2 independent experiments with 3 and 2 mice per group.