Background: Black and Hispanic children with B-acute lymphoblastic leukemia (B-ALL) experience worse outcomes compared to their non-Hispanic white (NHW) counterparts. Immune-based therapies have improved the outcomes of children with B-ALL, however, impact of racial/ethnic background on immune microenvironment is less studied. Methods: BM from 61 children with newly diagnosed B-ALL(Hispanic = 21, Black = 17, NHW = 23) was obtained via the Aflac Biorepository. High-dimensional analysis was performed utilizing single cell mass cytometry with 61 markers to characterize T, NK and myeloid cells. Data was analyzed using Cytobank and high-dimensional visualization platforms such as ViSNE. Clinical data including self-reported race/ethnicity and NCI-risk classification were obtained for all samples. Results: Multi-dimensional analysis was carried out for each cell population to dissect race/ethnicity-associated differences. ViSNE clustering of NK cells identified 3 different NK populations, including a distinct population of mature CD57+ NK cells with Tbet^hi, HLADR^hi, granzymeB^hi, CD27- phenotype. The distribution of NK subsets was highly impacted by race/ethnicity. Hispanic (H) patients had higher proportions of CD57+ mature NK cells when compared with other groups, (40 ± 4% vs 33 ± 2%; p = 0.03) with pronounced differences apparent within standard risk (SR) patients. H-SR had higher proportion of CD57+ NK cells compared to other SR patients (mean H-SR 43.4 ± 5.87% vs 26.3 ± 2.87% p = 0.0049). ViSNE clustering of myeloid cells identified 5 clusters based on patterns of cell surface markers, including a distinct CD11c+CD16+DR^hi inflammatory/non-classical myeloid population. Further analysis showed that NHW-SR patients have significantly lower proportions of CD16+DR+ myeloid cells compared to Hispanic, Black and NHW-HR patients (mean NHW-SR 3.67 ± 2.56% vs Others 10.8 ± 7.87% p = 0.0394). Notably, a phenotypically similar population has recently been implicated in leukemic progression in preclinical models (Witkowski et al, Cancer Cell 2020). In contrast to innate cells, T cell clusters were broadly comparable between different racial/ethnic groups. Conclusions: These studies provide detailed single-cell proteomic analysis and highlight the impact of racial/ethnic background on immune microenvironment in pediatric B-ALL. Our data identify differences in innate immunity with enrichment of high-risk immune-populations in Hispanic and Black children and depletion of inflammatory myeloid populations in NHW-SR children with B-ALL. These variations may contribute to the observed differences in outcomes and may impact application of immune therapies in racial/ethnic groups.