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Background Aims: The current approach for preventing hemolysis of red blood cells (RBCs) in major ABO-incompatible bone marrow (BM) grafts after infusion is to deplete RBCs from BM products before transplantation. Traditionally, manual density separation (MDS) using Ficoll-Hypaque (Cytiva Sweden AB, Uppsala, Sweden has been used to accomplish RBC depletion. This process yields good CD34 + cell recovery, but it requires open manipulation and is labor-intensive and time-consuming. We hypothesized that an alternative automated method using Haemonetics Cell Saver 5+ (Haemonetics Corporation, Boston, MA, USA) would offer equivalent RBC depletion and CD34 + cell recovery. Small marrow volumes from pediatric donors can be processed using Cell Saver (CS) without adding the third-party RBCs necessary for other automated methods., Methods: This retrospective analysis comprised data from 58 allogeneic BM grafts. RBC depletion and CD34 + cell recovery from BM using MDS (35 grafts) were compared with CS (14 grafts). Nine products underwent RBC depletion using CS with Ficoll (CS-F) when RBC volume was less than 125 mL., Results: Linear regression analysis of log transformation of CD34 + cell recovery adjusted for log transformation of both baseline CD34 + cell content and baseline total volume showed no significant difference between MDS and CS (estimated coefficient, -0.121, P = 0.096). All products contained an RBC volume of less than 0.25 mL/kg post-processing. CD34 + cell recovery with CS-F was comparable to MDS and CS and suitable for pediatric recipients of allogeneic hematopoietic cell transplantation., Conclusions: We provide evidence that an automated method using Haemonetics Cell Saver 5+ achieves RBC depletion and CD34 + cell recovery comparable to MDS when adjusting for baseline factors., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)