<p>Background</p><p>Although advances in immunotherapy have increased awareness of the importance of immunity in malignancy, it has not yet been fully investigated in pediatric myeloid malignancies. In this study, we conducted mass cytometric analysis on samples from pediatric patients with myeloid malignancies.</p><p>Method</p><p>Cryopreserved bone marrow samples from seven patients with myeloid malignancies and two controls were used for mass cytometric analysis. Various immune cell subsets and leukemic cells were identified using metal-conjugated antibodies against 28 surface antigens. The multidimensional data underwent clustering after dimensionality reduction.</p><p>Results</p><p>Firstly, all types of acute myeloid leukemia (AML) cells, including AML-M0, M2, M3, M5, M7, and AML with myelodysplasia-related changes, were clearly identified among the various immune cell subsets. In addition to the identification of leukemic cells, a detailed evaluation of immune cell subsets was possible. Mass cytometric analysis was also useful for assessing changes in the immune environment over the course of treatment and for the identification of lineage switches in mixed-phenotype acute leukemia. Finally, we tested the applicability of mass cytometry for the evaluation of measurable residual disease using serial dilution experiments. Although the barcoding system requires optimization, leukemic cells were clearly separated from normal cells across all AML types.</p><p>Conclusions</p><p></p><p>Mass cytometry may be useful for evaluating leukemic cells and the immune environment in pediatric myeloid malignancies.</p>