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Background: Bone marrow transplantation (BMT) is being routinely done at the Army Hospital for the past 15 years. Our experience with Stem Cell Transplantation and clinical trial on stem cell efficacy, our Centre had brought out the issue of trial deviates (<2 × 108 mononuclear cells [MNCs]) based on low yield of MNCs. PubMed search did not reveal any comparative data of analyzers versus manual counts. Thus, we decided to investigate the cause for the discrepancy calculated on 3-part and 5-part analyzers and manual estimation. This study has institutional relevance for hospitals involved in hematopoietic stem cell transplantation, therapy, and research. Introduction: MNCs are widely being used as a morphologic analog of precursor cells in hematopoietic stem cell transplantation and BMT units. The aim of the present study was, therefore, to evaluate the validity of the automated blood cell counter with gold standard of a manual count. Methods: The samples of peripheral blood (n = 80) and bone marrow (n = 80) were included in the study. Informed consent was obtained, and approval was taken from the IEC (Institutional Ethics Committee), Army Hospital Research and Referral, New Delhi, India. Results: Evaluation of MNC with 5-part hematology analyzer and gold standard manual count shows the correlation in the group of total leukocyte count samples: <4000, 4000–12,000, and 12,000–20,000 with no significant difference and >20,000 showing significant difference. Conclusion: The good correlation of 5-part hematology analyzer with manual gold standard method of MNC count is indicating equivalent performance. MNC can be performed by 5-part with nonsignificant difference and can be used as good as manual count.