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Background: Hematology analyzers may ineffectively recognize abnormal cells, and manual differential counts may be imprecise for leukopenic samples. We evaluated the efficacy of the Hematoflow method for determining the leukocyte differential in leukopenic samples and compared this method with the manual differential method. Methods: We selected 249 blood samples from 167 patients with leukopenia (WBC counts, 500–2,000/μL) for analysis in this study. The EDTA-anticoagulated blood samples were analyzed using an automatic blood cell counter (DxH800; Beckman Coulter, USA) and flow cytometry (FC 500; Beckman Coulter) by using Cytodiff reagent and analysis software (Beckman Coulter). Hematoflow results were selected or calculated from DxH800 and Cytodiff results. Two trained pathologists performed a manual differential count by counting 50–100 cells. Results: The precision of the Hematoflow method was superior to that of the manual method in counting 5 leukocyte subpopulations, immature granulocytes (IGs), and blasts. Blasts were detected in all 45 cases (100%) by Hematoflow. The correlation of the Cytodiff blast count to the reference count was high (r = 0.8325). For all other cell populations, the correlation of the Hematoflow results with the reference count was stronger than that of the other manual counts with the reference count. Conclusions: The Hematoflow differential counting method is more reproducible and sensitive than manual counting, and is relatively easy to perform. In particular, this method detected leukemic blasts more sensitively than manual differential counts. The Hematoflow method is a very useful supplement to automated cell counting.