Background: Flow cytometry (FC) has proven its utility in scrutinizing AB antigen expression in red blood cells (RBCs), cooperating with serological tests for accurate blood group typing. However, technical difficulties may impair the characterization of weak ABO subtypes when background noises appear at non-negligible levels. Study design and methods: We sought to establish an FC method that could prevent antibody-induced hemagglutination and an increase in cellular autofluorescence, two major issues inherent to RBC-FC analysis of AB expression.We optimized fixatives, multicolor-staining protocols, and sequential gating strategies. Blood samples from weak ABO subtype cases, B m and A el , were analyzed with the established protocol. Results:The optimized mixture of glutaraldehyde and formaldehyde successfully generated fixed RBCs resistant to agglutination while maintaining low autofluorescence. These features allowed co-staining of leukocyte-and erythrocyte-markers, which enabled sequential gating strategies facilitating the precise AB antigen analysis in purely single RBCs with minimum background noises. By the established FC analysis, we could detect in the B m sample a small RBC population exhibiting weak B antigen expression. The assay also proved it feasible to identify a small population (0.04%) of RBCs weakly expressing the A antigen in the A el sample confirmed as harboring a rare c.816dupG ABO variant allele. Conclusion:The RBC-FC analysis described here allows the detection of AB antigens weakly expressed in RBCs while achieving minimum background noise levels in negative control samples. Overall, the modified protocol provides a quick and reliable assay valuable in transfusion medicine and is potentially applicable to the characterization of rare weak ABO variants.