AbstractHomologous disruption of expression of signal transducer and activator of transcription 5a (STAT5a) and STAT5b (STAT5ab–/–) in mice results in hematopoietic stem cells (HSCs) that can engraft irradiated hosts alone but are noncompetitive against wild-type HSCs. To explore mechanisms for this phenotype, we crossed the STAT5 mutations onto an HW80 background congenic to the original C57BL/6 that differs in a small chromosome 7 genomic locus. We previously demonstrated that C57BL/6 or HW80 background STAT5ab–/– bone marrow (BM) cells showed equal repopulating function either competitively or noncompetitively in irradiated hosts. However, one intraperitoneal injection of wild-type green fluorescent protein (GFP) transgenic BM cells into unconditioned newborn STAT5ab–/– recipients of either background was sufficient for high-level donor engraftment. Furthermore, haploinsufficiency of STAT5 (STAT5ab+/–) allowed improved engraftment over wild-type recipients, indicating a dose-dependent requirement for STAT5 activation. In reciprocal experiments, STAT5ab–/– BM was transplanted into nonirradiated W/Wv hosts. In these mice, C57BL/6 STAT5ab–/– BM cells were 10-fold more defective in long-term engraftment than control wild-type BM cells and HW80 STAT5ab–/– BM cells were 5- to 10-fold more defective than C57BL/6 STAT5ab–/– BM cells. Therefore, we conclude that STAT5 plays a critical role during steady-state HSC engraftment and a chromosome 7 modifier locus regulates this activity.