Reprints and permissions information is available at www.nature.com/reprints. Data availability All sequencing data from this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) and are accessible through the GEO Series accession code GSE122713. All other relevant data are available from the corresponding author on request.

Graphical Abstract Highlights d Technology for high-throughput single-cell RNA sequencing and genotyping d Variable cell-type composition of AML correlates to genetics and outcome d Primitive AML cells aberrantly co-express stemness and myeloid priming genes d Differentiated AML cells express immunomodulatory factors and suppress T cells In BriefA combination of transcriptomics and mutational analyses in single cells from acute myeloid leukemia patients reveals the existence of distinct functional subsets and their associated drivers. SUMMARYAcute myeloid leukemia (AML) is a heterogeneous disease that resides within a complex microenvironment, complicating efforts to understand how different cell types contribute to disease progression. We combined single-cell RNA sequencing and genotyping to profile 38,410 cells from 40 bone marrow aspirates, including 16 AML patients and five healthy donors. We then applied a machine learning classifier to distinguish a spectrum of malignant cell types whose abundances varied between patients and between subclones in the same tumor. Cell type compositions correlated with prototypic genetic lesions, including an association of FLT3-ITD with abundant progenitor-like cells. Primitive AML cells exhibited dysregulated transcriptional programs with co-expression of stemness and myeloid priming genes and had prognostic significance. Differentiated monocyte-like AML cells expressed diverse immunomodulatory genes and suppressed T cell activity in vitro. In conclusion, we provide single-cell technologies and an atlas of AML cell states, regulators, and markers with implications for precision medicine and immune therapies.