<p>Autophagy induction has recently emerged as a mechanism of resistance to FLT3 inhibitors (FLT3i) in patients with <i>FLT3</i>-ITD mutant acute myeloid leukemia (AML). Here, we assessed the molecular mechanisms of autophagy inhibition associated with FLT3i and its impact on cell survival and pharmacological resistance. In <i>FLT3</i>-ITD AML cell lines (MOLM13 and MV4-11), treatment with first- and second-generation FLT3i (midostaurin and quizartinib, respectively) induced autophagy. Combining FLT3i with autophagy inhibitors further decreased cell viability and increased cell apoptosis in both cell lines and in primary patient samples. Label-free quantification proteomics of MOLM13 cells revealed that RFC4 (Replication Factor C Subunit 4), an autophagy regulator linked to increased chemosensitivity, and GATD3/C21orf33 (Glutamine Amidotransferase Class 1 Domain Containing 3) proteins were upregulated only in the combined group, while 11 proteins mostly associated with chemoresistance were downregulated. In vivo, the combination of midostaurin and autophagy inhibition improved overall survival in MOLM13-transplanted mice. <i>ATG5</i>- (Autophagy Related 5) and <i>ATG7</i>-knockdown (Autophagy Related 7) increased sensitivity to first- and second-generation FLT3i in MOLM13 cells. To investigate the potential of autophagy inhibition in overcoming FLT3i resistance, we generated MV4-11 cells resistant to quizartinib (MV4-11QR). The resistant cell line exhibited higher basal levels of autophagy compared to the parental cell line. The combination of quizartinib and chloroquine demonstrated a synergistic effect in MV4-11QR cells and this effect was associated with greater inhibition of the FLT3 receptor compared to the monotherapies. Therefore, combining FLT3i with autophagy inhibition enhances the FLT3i antileukemic efficacy and overcomes pharmacological resistance.</p>