HSCs lacking Nfix are deficient in hematopoietic repopulating potential. (A) Schematic of transplant strategy to assess hematopoietic repopulating potential of HSC. At 24 hours posttransduction with either control- or Nfix-shRNAs, 5000 CD45.2 LSK cells (test cells) were transplanted along with 5000 CD45.1 mock-transduced LSK cells (competitor cells) into lethally irradiated CD45.1/CD45.2 recipient mice. Residual uninjected cells were analyzed via flow cytometry to confirm injection of an equal ratio of test and competitor cells. (B) LSK cells were transduced with either control- or Nfix-shRNAs and analyzed after 3 days of serum-free liquid culture for %mCherry⁺ cells by flow cytometry. Consistently, 75% to 80% of cells were mCherry⁺ at 3 days postinfection. Results represent mean ± standard deviation from 3 independent experiments. (C) The peripheral blood of recipient mice was analyzed every 4 weeks posttransplant by flow cytometry for contribution from CD45.2⁺ test cells. LSK cells infected independently with 2 shRNAs targeting Nfix displayed a dramatic decrease in repopulating potential, relative to test cells transduced with control-shRNAs, as early as 4 weeks posttransplant. Each point represents the mean ± standard error of the mean of 3 independent experiments with >3 recipient mice in each experiment. (D) No major differences were seen in the distribution of cell surface markers representing select blood lineages in the CD45.2⁺ mCherry⁺ peripheral blood compartment of recipients transplanted with LSK cells transduced with control- or Nfix-shRNAs 16 weeks posttransplantion. Results represent mean ± standard error of the mean of 3 independent experiments with >3 recipient mice in each experiment. (E) Schematic of transplant strategy to assess hematopoietic repopulating potential of Nfix^Δ/Δ HSC. At 24 hours posttransduction with Cre recombinase, 5000 CD45.2 Nfix^fl/fl or Nfix^+/+ LSK cells (test cells) were transplanted along with 5000 CD45.1 mock-transduced LSK cells (competitor cells) into lethally irradiated CD45.1/CD45.2 recipient mice. Residual cells were cultured under serum-free conditions and analyzed 5 days posttransduction by polymerase chain reaction for the presence of floxed, deleted, and wild-type alleles (F). (G) The peripheral blood of recipient mice analyzed at 4 and 7 weeks posttransplant with Nfix^fl/fl or Nfix^+/+ test cells transduced with Cre recombinase. (H) This experiment is representative of 2 independent experiments. No major differences were seen in the distribution of cell surface markers representing select blood lineages in the CD45.2⁺ peripheral blood compartment of recipients transplanted with Nfix^fl/fl or Nfix^+/+ test cells transduced with Cre recombinase 7 weeks posttransplant. P values were considered statistically significant when *P < .05, **P < .01, and ***P < .001.