HSPCs lacking Nfix are diminished in the bone marrow of recipient mice. (A) Representative analysis of WBM collected from recipient mice transplanted 16 weeks prior with CD45.2 LSK cells transduced with either control- or Nfix-shRNAs. Cells were stained with CD45.1, CD45.2, lineage markers, Sca-1, c-Kit, CD48, and CD150, and were analyzed via flow cytometry for the persistence of mCherry⁺ cells in the CD45.2⁺ compartment of the LSK and LSK CD48⁻CD150⁺ marrow fractions. A nonmanipulated control animal is shown for comparison. (B) Analysis of frequency of CD45.2⁺ cells and mCherry⁺ cells within the CD45.2⁺ compartment of the WBM, LSK cells, and Lineage⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ cells of recipient mice transplanted 16 weeks prior, as previously described. Results represent mean ± standard deviation from 2 independent experiments. (C) Representative flow cytometry analysis of frequency of CD45.2⁺ cells within the LSK and LSK CD48⁻CD150⁺ bone marrow compartment of recipient mouse transplanted 7 weeks prior with CD45.2⁺Nfix^fl/fl or Nfix^+/+ LSK cells transduced with Cre recombinase. (D) Analysis of frequency of CD45.2⁺ cells within the LSK and LSK CD48⁻CD150⁺ bone marrow compartment of individual recipient mice transplanted 7 weeks prior with CD45.2⁺Nfix^fl/fl or Nfix^+/+ LSK cells transduced with Cre recombinase. CD45.2⁺ cells were isolated by FACS from c-Kit-enriched bone marrow collected from individual mice and analyzed by genomic polymerase chain reaction for the presence of deleted and floxed Nfix alleles. The floxed Nfix allele was undetectable in all recipients of Cre-transduced Nfix^fl/fl LSK cells, except recipient E-labeled mouse. Recipient E-labeled mouse was the only recipient of Cre-transduced Nfix^fl/fl LSK cells that contained a high percentage of CD45.2⁺ cells in all 3 compartments examined (total bone marrow, LSK, and LSK CD48⁻CD150⁺). P values were considered statistically significant when *P < .05, **P < .01, and ***P < .001.