Hematopoietic stem cells (HSCs) express Mdr1a/1b and Bcrp1/Abcg2, which are members of the ATP binding cassette transporter family. Mice lacking both Mdr1-type genes (Mdr1a and Mdr1b) or Bcrp1 had normal hematopoietic development, but it has been unclear whether Mdr1a/1b and Bcrp1 play redundant roles in hematopoiesis. We generated a mouse model lacking both Mdr1a/1b and Bcrp1 expression (M-/-B-/-). The M-/-B-/- mice had normal numbers of peripheral blood cells, bone marrow colony-forming cells (CFCs) and colony-forming units-spleen (CFU-S), and demonstrated normal hematopoietic development. There was a near total elimination of side population (SP) cells in the bone marrow of M-/-B-/- mice compared to M+/+B-/- mice, primarily in the subpopulation lacking other HSC markers, which indicated that Mdr1a/1b was responsible for a small portion of SP cells that were mainly mature cells. Hematopoietic progenitor cells from the bone marrow of M-/-B-/- mice were more sensitive to mitoxantrone in vitro compared to either M-/-B+/+ or M+/+B-/- mice, suggesting that Mdr1a/1b and Bcrp1 may provide additive protection to HSCs against genotoxic agents. These studies demonstrate the lack of functional redundancy between these transporters for HSC development and further clarify their contributing role to the SP phenotype in HSCs and to intrinsic drug resistance within hematopoietic progenitor cells.