Differentiation of ABO mosaics from chimeras is performed using flow cytometry (FCM) analysis. Although mosaics and chimeras have been distinguished by presence or absence of clear resolution using FCM analysis, the lack of quantitative metrics and definitive criteria for this differentiation has made some cases difficult to differentiate. In this study, therefore, we attempted to establish a definitive and quantitative criterion for this differentiation. When FCM histogram gates for group "A" or "B" antigen-negative and -positive red blood cells (RBCs) were set such that group O RBCs were classified as 99 percent negative and group A or B RBCs as 99 percent positive, the percentages of RBCs in the middle region of six chimeras and 23 mosaics (12 A mosaics and 11 B mosaics) were 0.1-0.6 percent and 7.0-19.0 percent, respectively. This results suggested that ABO mosaics and chimeras can be unambiguously differentiated when the cutoff point of the intermediate region is set to 1 percent.