B220⁺Gr1^–c-Kit⁺cells contain leukemia-initiating cells. (A) Representative flow cytometric profiles of c-Kit in the B220⁺Gr1^–, B220⁺Gr1⁺, and B220^–Gr1⁺ fractions of leukemic 3q21q26 (left) and 3q21q26::Gata2^+/− (right) mouse bone marrows. Note that the B220⁺Gr1^– fraction is predominant in the 3q21q26::Gata2^+/− mice in right panel. (B) Percentages of c-Kit⁺ cells in the B220⁺Gr1^–, B220⁺Gr1⁺, and B220^–Gr1⁺ fractions in the bone marrow of the leukemic 3q21q26 mice (n = 35) and the B220⁺Gr1^– fraction in the leukemic 3q21q26::Gata2^+/− mice (n = 15). Parameters for the box plots are defined in the Statistical analysis paragraph of “Methods.” (C) Wright-Giemsa staining of leukemic cells in the B220⁺Gr1^–, B220⁺Gr1⁺, and B220^–Gr1⁺ fractions from the leukemic 3q21q26 and B220⁺Gr1^– fraction from the leukemic 3q21q26::Gata2^+/− mice (c-Kit⁺ [top line] and c-Kit⁻ [bottom line]). (D) Colony-forming ability of B220⁺Gr1^–c-Kit⁺, B220⁺Gr1⁺c-Kit⁺, B220^–Gr1⁺c-Kit⁺, B220⁺Gr1^–c-Kit^–, B220⁺Gr1⁺c-Kit^– and B220^–Gr1⁺c-Kit^– populations from the leukemic 3q21q26 mice (n = 6). Representative colony morphologies from the B220⁺Gr1^–c-Kit⁺, B220⁺Gr1⁺c-Kit⁺, and B220^–Gr1⁺c-Kit⁺ populations are shown in the right panels. (E) Kaplan-Meier survival curves of mice receiving B220⁺Gr1^–c-Kit⁺ (n = 16; median, 56 days), B220⁺Gr1⁺c-Kit⁺ (n = 8; median, not determined), and B220^–Gr1⁺c-Kit⁺ (n = 6; median, not determined) cells. (F) Representative flow cytometric patterns of leukemic cells in donor mice and donor-derived (CD45.1 homozygous or CD45.1/CD45.2 heterozygous) leukemic cells in recipient mice receiving B220⁺Gr1^–c-Kit⁺ cells. (G) Relative expression levels of human EVI1 messenger RNA (mRNA) (left panel), total (human + mouse) EVI1 mRNA (middle panel), and mouse endogenous Gata2 mRNA (right panel) in B220⁺Gr1^–c-Kit⁺ (n = 6), B220⁺Gr1⁺c-Kit⁺ (n = 5), B220^–Gr1⁺c-Kit⁺ (n = 4), B220⁺Gr1^–c-Kit^– (n = 4), B220⁺Gr1⁺c-Kit^– (n = 3), and B220^–Gr1⁺c-Kit^– (n = 3) populations. Bar graphs represent mean ± standard deviation (SD). *, P < .05; **, P < .01.