Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) has poor outcomes. FLT3-ITD drives constitutive and aberrant FLT3 signaling, activating STAT5 and upregulating the downstream oncogenic serine/threonine kinase Pim-1. FLT3 inhibitors have limited clinical efficacy. We previously showed that concurrent Pim and FLT3 inhibition increases apoptosis induction in FLT3-ITD-expressing cells through post-translational downregulation of Mcl-1. Here we further elucidate the mechanisms of action of this dual targeting strategy. Protein expression and turnover, cytotoxicity and apoptosis were measured in FLT3-ITD-expressing cell lines and AML blasts treated with FLT3 inhibitor gilteritinib and/or Pim inhibitors AZD1208 or TP-3654. Pim and FLT3 inhibitor co-treatment decreased c-Myc protein, prior to Mcl-1, increased turnover of both proteins, rescued by proteasome inhibition, dephosphorylated (activated) GSK-3β, and increased apoptosis and in vivo efficacy. GSK-3β inhibition prevented c-Myc and Mcl-1 downregulation and apoptosis. Pim and FLT3 inhibitor co-treatment of Ba/F3-ITD cells infected with T58A c-Myc or S159A Mcl-1 plasmids, preventing phosphorylation at these sites, did not downregulate these proteins, increase their turnover or induce apoptosis, consistent with GSK-3β activation and c-Myc T58 and Mcl-1 S159 phosphorylation as the mechanism of combination treatment. These data further support GSK-3β activation as a therapeutic strategy in FLT3-ITD AML.