Using a cohort of C57BL/6 (B6) x (NZB x B6)F1 backcross male mice bearing the Yaa (Y-linked autoimmune acceleration) mutation, we mapped and characterized the NZB-derived susceptibility loci predisposing to the development of autoimmune hemolytic anemia (AHA). Our analysis identified 2 major loci on NZB chromosome 7 and chromosome 1 linked with Coombs antierythrocyte autoantibody production, and their contributions were confirmed by the analysis of B6.Yaa mice (B6 mice bearing the Yaa mutation) congenic for each NZB-derived susceptibility interval. A newly identified Aia3 (autoimmune anemia 3) locus present on NZB chromosome 7 selectively regulated Coombs antibody responses, while the second locus, directly overlapping with Nba2 (NZB autoimmunity 2) on chromosome 1, promoted the development of AHA, likely as part of its effect on overall production of lupus autoantibodies. A higher incidence of Coombs antibody production in B6.Aia3 congenic mice (B6 mice bearing the NZB-Aia3 locus) than B6.Nba2 mice (B6 mice bearing the NZB-Nba2 locus) indicated a major role for Aia3 in AHA. Notably, lack of expansion of B1 cells in B6.Aia3 congenic mice argued against the involvement of this subset in AHA. Finally, our analysis of BC mice also demonstrated the presence of a B6-derived H2-linked locus on chromosome 17 that apparently regulated the production of Coombs antibodies as a result of its overall autoimmune promoting effect.