Chronic myeloid leukemia (CML) is a myeloproliferative disease derived from hematopoietic stem cells (HSCs) that harbor Philadelphia chromosome (Ph chromosome). In clinic, leukemia stem cells (LSCs) in CML are insensitive to the treatment with tyrosine kinase inhibitors, and are responsible for disease relapse. However, the molecular mechanisms for maintaining LSCs survival remain elusive.CML patient-derived cell lines and BCR-ABL-induced CML mouse model were used to explore the role of YBX1 in regulating the survival of CML LSCs. Bone marrow transduction and transplantation, and colony-forming unit assay were used to investigate LSC function. The underlying mechanism of how YBX1 regulates LSCs survival were assessed using flow cytometry, RNA sequencing, western blot, RNA decay assay, co-immunoprecipitation and RNA immunoprecipitation.Here we show that RNA-binding protein YBX1 plays an important role in regulating survival of CML LSCs. We find that YBX1 expression is significantly increased in CML cells, and confirm that YBX1 is required for maintaining survival of LSCs. Deletion of YBX1 impairs the propagation of CML through blocking cell proliferation and inducing apoptosis of LSCs. Mechanistically, we find that YBX1 regulates expression of apoptotic associated genes. YBX1 cooperates with RNA m6A reader IGF2BPs to stabilize YWHAZ transcript in an m6A-dependent manner, and loss of YBX1 decreases YWHAZ expression by accelerating mRNA decay. Restoration of YWHAZ efficiently rescues the defects of YBX1-deficient CML cells.Our findings reveal a critical role of YBX1 in maintaining survival of CML LSCs, which provides a rationale for targeting YBX1 in CML treatment.© 2022. Springer Nature Switzerland AG.