All thymic progenitor activity resides within the lineage⁻Flk2⁺CD27⁺ subset, which includes MPPs and CLPs. (A) BM was separated into fractions positive and negative for mature lineages (CD3, CD4, CD8, CD11c, Mac-1, Gr-1, B220, CD19, Ter119, and NK1.1). The entirety of each population (9 × 10⁵ lineage⁺, 3 × 10⁵ lineage⁻) was injected intravenously into 5 sublethally irradiated CD45-congenic recipients. Thymuses were analyzed for donor chimerism 14 days later, and all thymic progenitor activity was found in the lineage⁻ subset. Each flow cytometry plot is representative of 5 recipients. Arrows in top panels indicate gating hierarchy. (B) Thymic chimerism of sublethally irradiated CD45 congenic mice 14 days after injection of 10⁵ lineage⁻ Flk2⁺CD27⁺ cells, or all remaining (3 × 10⁶) lineage⁻ non-Flk2⁺CD27⁺ cells. All thymic progenitor activity was found within the Flk2⁺CD27⁺ subset. Data are representative of 4 independent experiments. The average donor Thy1.1⁺ chimerism of the recipients of Flk2⁺CD27⁺ and non-Flk2⁺CD27⁺ populations were 1.9% and 0.0008%, respectively. (C) Representative BM stain used for sorting. BM was depleted of mature lineages using antibodies to CD3, CD4, CD8, B220, Mac-1, Gr-1, and Ter119 and stained for residual lineage⁺ cells. CD19⁺ and CD11c⁺ cells were excluded in separate channels. A total of 2.5 × 10⁶ events were collected. The lineage⁻ Flk2⁺CD27⁺ population was divided into IL-7R⁻ MPP (c-Kit^hi and Sca-1^hi) and IL-7R⁺ CLP, which had uniform low levels of c-Kit and Sca-1.