Single-cell mass cytometry (SCMC) combines features of traditional flow cytometry (i.e.,fluorescence-activated cell sorting) with mass spectrometry,making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms.In this study,we optimized SCMC to analyze hemoeytes of the Drosophila innate immune system.We used metal-conjugated antibodies (against cell surface antigens H2,H3,H18,L1,L4,and P1,and intracellular antigens 3A5 and L2) and anti-IgM (against cell surface antigen L6) to detect the levels of antigens,while anti-GFP was used to detect crystal cells in the immune-induced sam-ples.We investigated the antigen expression profile of single cells and hemocyte populations in naive states,in immune-induced states,in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase (hopTum) and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1[l(3)mbn1],as well as in stem cell maintenance-defective hdcΔ84 mutant larvae.Multidimensional analysis enabled the discrimination of the functionally dif-ferent major hemocyte subsets for lamellocytes,plasmatocytes,and crystal cells,and delineated the unique immunophenotype of Drosophila mutants.We have identified subpopulations of L2 +/P1 +and L2+/L4+/P1+ transitional phenotype cells in the tumorous strains l(3)mbn1 and hopTum,respectively,and a subpopulation of L4+/P1+ cells upon immune induction.Our resultsdemonstrated for the first time that SCMC,combined with multidimensional bioinformatic analy-sis,represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immu-nity of Drosophila.