Long-term thymic differentiation of donor progenitors in IT-injected mice does not result in the engraftment of BM progenitors with secondary repopulating ability. (A) ZAP-70^−/− mice were injected intravenously or intrathymically with CD45.1⁺ WT lin⁻ BM cells (2 × 10⁵). Bone marrow was harvested 20 to 25 weeks after transplantation, and the absolute numbers of lineage-negative (negative for Ter119, B220, Mac-1, Gr-1, and CD3 staining) CD45.1⁺ cells in the BM were assessed in mice who received an intravenous (IV) transplant (n = 5) and mice that received an IT transplant (n = 4). Absolute numbers of engrafted lin⁻/Sca-1⁺/c-Kit⁺ cells were also determined. *P = .02. (B) Secondary repopulation ability was assessed by transplanting T cell–depleted BM (9 × 10⁶) from these intravenously and intrathymically reconstituted ZAP-70^−/− mice (25 weeks after transplant) into lethally irradiated CD45.2⁺ ZAP-70^−/− mice (9 Gy [900 rad]). As a control, lethally irradiated CD45.2⁺ ZAP-70^−/− mice received a transplant with CD45.1⁺ T cell-depleted BM (9 × 10⁶) from WT mice. The presence of CD45.1⁺ cells in the spleen was assessed 13 weeks later. Representative dot plots showing the percentages of splenic CD45.1⁺ donor B/T lymphocytes are presented. (C) The relative levels of CD45.1⁺ donor cells in the thymi of secondary reconstituted mice are shown in representative dot plots. Results represent data obtained in 3 independent mice.