Background: Lymphocyte subset analysis is essential to evaluate the engraftment status in hematopoietic stem cell transplantation (HSCT). Automated gating tools are widely used for flow cytometry analysis. Unlike healthy individuals, different cell populations and aberrant expressions may occur in HSCT samples. In the present study, we evaluated the applicability of automated gating in HSCT recipients by comparing it to expertbased manual gating. Methods: Lymphocyte subset was performed using Beckman Coulter Navios (Beckman Coulter, USA) flow cytometry. Data files from 22 patients with hematologic malignancies were analyzed in parallel by manual gating and automated gating using Navios Tetra software. Quality control results and reproducibility were evaluated using IMMUNO-TROL controls. Results: Spearman rank correlation coefficients between the two gating methods were > 0.970 in all cell populations except CD8+ T cells. CD8+ T cell counts via automated gating were higher than those of manual gating in all cases due to the T cell populations with reduced CD8 expression. Automated gating program failed to identify CD4+CD8+ double-positive T cell population. Moreover, it excluded certain lymphocytes with low forward scatter (FSC) and high side scatter (SSC). Furthermore, two HSCT recipients revealed a high percentage of CD56−CD16+ NK cells, we found the need to add CD16 reagent to the Navios system. All coefficients of variation were < 10% except for CD56+ NK cells via automated gating. Conclusions: Manual gating confirmation via flow cytometry histogram is necessary to identify the aberrant phenotypes and unexpected cell populations in HSCT recipients.