G protein-coupled receptor kinases (GRKs) are critically involved in immune response through regulation of cytokine receptors in mature leukocytes, but their role in hematopoiesis is largely unknown. Here, we demonstrate that GRK6 knockout (GRK6-/-) mice exhibit lymphocytopenia, loss of the hematopoietic stem cell (HSC) and multiple progenitor populations. GRK6 deficiency leads to compromised lymphoid differentiation, largely owing to the impairment of HSC self-renewal. Transcriptome and proteomic analysis suggest that GRK6 is involved in reactive oxygen species signaling. GRK6 could interact with DNA-PKcs (DNA-dependent protein kinase, catalytic subunit) and regulate its phosphorylation. Moreover, reactive oxygen species scavenger α-lipoic acid administration could partially rescue the loss of HSC in GRK6-/- mice. Our work demonstrates the importance of GRK6 in regulation of HSC self-renewal and reveals its potential role in participation of stress response.