Background: Hematogones are normal B-cell precursor which can be seen in different physiological and pathological conditions. Due to variation in B-cell acute lymphoblastic leukemia (B-ALL) blasts immunophenotyping and interference of hematogones in minimal residual disease (MRD) assessment, precise discrimination of hematogones is very crucial. The purpose of this study was to evaluate the expression pattern of surface markers in hematogones and compare them with lymphoblasts. Material and Methods: In this applied study, flow cytometric analysis was performed using Coulter FC-500 and MXP software in 4-color combination and 6 different tubes. In this study, 85 patients diagnosed with acute lymphoblastic leukemia were evaluated. Out of these patients, 45 were boys and 40 were girls. Patients aged from 1 to 15 years old. In addition, 27 bone marrow samples from other patients aged 4 to 18 years were included in this investigation. These samples had been obtained for other diagnostic purposes, such as immune thrombocytopenic purpura and juvenile idiopathic arthritis. Results: During flow cytometric analysis, hematogones showed expressions of CD19, CD20, CD22, CD10, CD45, CD81, CD123, CD9, CD34 (partial expression), and tdt (partial expression). In these patients, hematgones were negative for CD66c expression. Lymphoblastic cells were positive for CD19, CD20 (in some cases), CD22, CD10, CD45, CD81, CD123, CD58, CD9, CD66c, CD34 (in most cases), and TDT. CD81 mean fluorescence intensity (MFI) in hematogones was higher than that in lymphoblasts. (112. 5 (30-251) vs. 17. 5 (5-30); P<0. 0001) Conclusion: According to findings of this study, it seems that the use of CD81, CD58, CD123, CD66c, CD9, and CD81 MFI in combination with B-Cells associated markers can be very effective in differentiating hematogone from lymphoblast.