Steady state bone marrow (BM) is the preferred hematopoietic stem cell (HSC) source for gene therapy in sickle cell disease (SCD) due to the recognized risk of vaso-occlusive crisis during granulocyte colony–stimulating factor mobilization. We previously established clinically relevant HSC gene transfer in the rhesus model following transplantation of mobilized peripheral blood (PB) CD34⁺ cells transduced with lentiviral vectors. In this study, we examined steady state bone marrow (BM) in the rhesus competitive repopulation model and demonstrate similar gene marking in vitro and in vivo, as compared with mobilized PB CD34⁺ cells. We then evaluated PB and steady state BM in subjects with SCD and observed a higher frequency of CD34⁺ cells when compared with controls, likely due to enhanced hematopoiesis. However, CD34⁺ cell counts were reduced in both the PB and BM in patients treated with hydroxyurea, and hydroxyurea treatment strongly inhibited iPS cell generation from SCD subjects. Our data support that steady state BM is a useful HSC source for SCD gene therapy with similar transduction. The lower CD34⁺ percentages observed with hydroxyurea treatment warrants withholding hydroxyurea temporarily prior to harvesting HSCs. Our results are important for the design of gene targeting strategies for SCD.