Acute leukemias of ambiguous lineage (ALAL) are rare hematologic malignancies with poor outcomes. Retrospective studies have suggested that acute lymphoblastic leukemia (ALL) regimens are more effective than acute myeloid leukemia (AML) regimens. We retrospectively examined the effectiveness of the widely-used adult ALL regimen hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyperCVAD) as initial therapy in patients with ALAL at five academic institutions. Twenty-five patients were identified, including 23 with mixed phenotype acute leukemia (MPAL) and two with acute undifferentiated leukemia. Five of 8 tested (63%) had FLT3-ITD and 3 of 25 (12%) were Philadelphia chromosome-positive. The complete remission (CR) rate was 76%, with CR with incomplete count recovery (CRi) in an additional 8%, for an overall response rate of 84%. Median number of cycles to CR/CRi was 1. There were no deaths in the first 30 days. Of the 21 patients achieving CR or CRi, 14 (66%) proceeded to allogeneic hematopoietic stem cell transplantation. With a median follow-up time of 31.6 months, median overall survival for the entire cohort was not reached, and the estimated 2-year survival was 63%. HyperCVAD can be considered an effective and tolerable front-line regimen for patients with ALAL, and warrants further prospective study.