Therapeutic improvements are needed for patients with AML, particularly those who have relapsed or who have treatment-refractory (R/R) AML or newly diagnosed patients with poor prognostic factors. Alvocidib (DSP-2033), a potent cyclin-dependent kinase 9 inhibitor, has previously demonstrated promising clinical activity for the treatment of AML. In this multicenter, open-label, uncontrolled, 3+3 phase I study, we investigated the safety and tolerability of alvocidib administered in combination with either cytarabine and mitoxantrone (ACM) for R/R AML, or cytarabine/daunorubicin (A+7+3) for newly diagnosed AML. Alvocidib was administered to all patients as a 30-minute i.v. bolus (30 mg/m2 /d), followed by a continuous i.v. infusion over 4 hours on days 1-3 (60 mg/m2 /d). A total of 10 patients were enrolled: 6 received ACM (at 2 dose levels of cytarabine and mitoxantrone), and 4 received A+7+3. Alvocidib was tolerated and no dose-limiting toxicities were observed. All patients experienced adverse events, of which diarrhea was the most frequent (100%); hematologic events were also common. Alvocidib concentration peaked at the end of dosing (4.5 hours after start of administration), plasma accumulation after repeated dosing was minimal, and urinary excretion was negligible. The rate of complete remission/complete remission with incomplete hematologic recovery was 66.7% with the ACM regimen in R/R AML, including 4 complete remission (median duration 13.6 months) and 75% (3 complete remission) with the A+7+3 regimen. Further development of alvocidib in hematologic malignancies is warranted. The trial is registered with Clinicaltrials.gov: NCT03563560.