HSPCs lacking Nfix are established but fail to persist in the bone marrow of recipient mice. (A) Experimental schematic. Mice recipient of CD45.2⁺ were transplanted with 9000. CD45.1⁺ LSK cells transduced with control- or Nfix-shRNAs, and were then analyzed beginning at 5 days posttransplant for the presence of mCherry⁺ CD45.1⁺ bone marrow cells via flow cytometry. (B) At 5, 8, 12, and 20 days posttransplant, marrow from mice transplanted with CD45.1⁺ LSK cells transduced with either control- or Nfix-shRNAs was examined for the persistence of mCherry⁺ cells within both the CD45.1⁺ WBM and CD45.1⁺ LSK marrow. Results represent mean ± standard deviation from 2 independent experiments. (C) mCherry⁺ LSK cells were isolated via FACS from the marrow of pooled cohorts of mice transplanted 10 days prior with either control- or Nfix-shRNAs and then plated in semi-solid methycellulose-based medium supplemented with hematopoiesis promoting cytokines. Hematopoietic colonies were scored 10 days postplating. WBM from a cohort of nonmanipulated mice were used as a gating and sorting control. Results represent the mean ± standard deviation from 3 independent experiments. P values were considered statistically significant when *P < .05, **P < .01, and ***P < .001. CFU-C, colony forming unit-culture; PB, peripheral blood.