Mutations in the WT1 transcription factor (WT1) gene are found in ∼5% to 12% of adults with acute myeloid leukemia (AML) and are generally associated with a poor prognosis.1-8 Several studies have also reported frequent co-occurrence of FLT3-ITD in patients with mutated WT1, ranging from 23% to 75%.1,6,9,10 In some,1,10 but not all,5-7 analyses, patients with both WT1 mutations and FLT3-ITD had lower complete remission rates, higher relapse rates, and inferior overall survival than patients with mutated WT1, but no FLT3-ITD. Although WT1 mutations are common in adult AML, there are limited data describing whether they are initiating or late mutations in AML pathogenesis. Furthermore, the clonal architecture and complexity of this AML subtype are not well described. The utility of single-cell DNA sequencing has provided important biologic insights into other subtypes of AML, particularly with respect to drug resistance.11,12 In this study, we performed bulk targeted DNA sequencing of 96 newly diagnosed patients with AML with WT1 mutations, treated in Cancer and Leukemia Group B (CALGB) therapeutic trials, followed by single-cell DNA sequencing in a subset of patients to provide higher resolution and insight into both the initiating and clonal framework of mutations co-occurring with WT1. CALGB is now part of the Alliance for Clinical Trials in Oncology (Alliance).