Summary Myeloid malignancies, including acute myeloid leukemia (AML), arise from the expansion of hematopoietic stem/progenitor cells which acquire somatic mutations. Bulk molecular profiling suggests step-wise mutation acquisition, where mutant genes with high variant allele frequencies (VAFs) occur early in leukemogenesis and mutations with lower VAFs are thought to be acquired later 1 – 3 . Although bulk sequencing informs leukemia biology and prognostication, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate mutational order. To delineate the clonal framework of myeloid malignancies, we performed single cell mutational profiling on 146 samples from 123 patients. We found AML is dominated by a small number of clones, which frequently harbor co-occurring mutations in epigenetic regulators. Conversely, mutations in signaling genes often occur more than once in distinct subclones consistent with increasing clonal diversity. We next mapped clonal trajectories for each sample and uncovered mutation combinations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our studies of single cell clonal architecture provides novel insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

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