The Y-box-binding protein 1 (YBX1), as a member of RNA-binding proteins, has been implicated as critical regulator of cell survival in various solid tumors and acute myeloid leukemia (AML). However, the function of YBX1 in T-cell acute lymphoblastic leukemia (T-ALL) remains elusive. Here we found that YBX1 is upregulated in T-ALL patients, cell lines and NOTCH1-induced T-ALL mice. Furthermore, depletion of YBX1 dramatically reduced cell proliferation, induced cell apoptosis and G0/G1 phase arrest in vitro. Moreover, YBX1 depletion significantly decreased leukemia burden in the human T-ALL xenograft and NOTCH1-induced T-ALL mice model in vivo. Mechanistically, downregulation of YBX1 markedly inhibited the expression of total AKT, p-AKT, total ERK and p-ERK in T-ALL cells. Taken together, our results uncovered a critical role of YBX1 in the leukemogenesis of T-ALL, which may have great potential as a biomarker and therapeutic target in T-ALL.Copyright © 2023 American Society of Hematology.