Aged MPPs are inefficient at producing T-lineage cells in vivo and in vitro. (A) Two thousand sorted MPPs from 2-month-old or 18- to 24-month-old donors were injected into the thymi of nonirradiated recipients, and donor-derived DP progeny were analyzed 21 days later. Error bars represent 1 SEM (*P = .05). (B) Two hundred to 250 purified MPPs were plated onto OP9-DL1 stromal layers supplemented with IL-7 and FL, and numbers of T-lineage (Thy1⁺ CD25⁺) and myeloid lineage (Mac1⁺) progeny were assessed 8 to 10 days later. For bar graphs, error bars represent 1 SEM (*P < .01). (C) Limit-dilution analysis was performed in vitro by culture of purified 2-month-old and 18- to 24-month-old MPPs on OP9-DL1 stromal layers. Poisson statistics determined frequencies of lineage-competent cells (T lineage young: 1 in 19 [95% confidence interval 1 in 13-26], aged: 1 in 21 [1 in 15-29], P = .6; myeloid lineage young: 1 in 13 [1 in 9-17], aged: 1 in 10 [1 in 8-14], P = .3). Twenty-eight wells were analyzed for each cell dose.