Ski is critically required for HSC fitness in the absence of transplantation. (A) Representative fluorescence-activated cell sorter plots of E14.5 FL cells gated on live, single lineage^– cells. All scales are log₁₀. LSK: lineage^–Sca-1⁺c-Kit⁺ (supplemental Figure 2C-D). (B) For HSC transplantation controls, E14.5 FL cells were transplanted in the absence of competitors (5-6 mice per group), (C) for competitive Ski^−/− HSC transplantation, E14.5 FL cells were transplanted at a 1:1 ratio to competitors (6 mice per group), (D) for dosage dependency, E14.5 FL cells were transplanted at a 4:1 ratio to competitors (6 mice per group), and (E) for competitive Skil^−/− HSC transplantation, adult whole Skil^−/− bone marrow cells were transplanted at a 1:1 ratio to competitors (6 mice per group), and recipients in panels B-E were analyzed for the percentage of donor contribution to peripheral blood chimerism and total number of SLAM HSCs. (F) Southern blot of Ski^+/+, Ski^+/− or Ski^−/− ES cells used in blastocyst complementation assays confirms genotype. (G) A representative glucose phosphate isomerase assay used to quantify chimerism in blastocyst complementation assays. (H) A heatmap representation of tissue-specific chimerism in progeny from a blastocyst complementation assay with C57BL/6 mouse strain blastocysts complemented with 1 Ski^+/+, 1 Ski^+/−, or 2 independent Ski^−/− ES cell clones derived from a 129P3 mouse (5 mice per clone). (B-E) data are displayed as mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.