Simple SummaryRhabdomyosarcoma (RMS) is a devastating pediatric sarcoma. In particular, patients with relapsed disease have very poor survival outcomes. However, there has not been any significant change in therapy options for the last three or four decades, and the mechanisms underlying treatment failures remain poorly understood. In this study, we showed that two genes,MYCandYBX1, are essential for the viability of chemotherapy-tolerant cells that lead to resistance. We also demonstrated the mutual regulation of MYC and YBX1 function as a novel mechanism of therapy resistance in RMS. Targeting MYC, YBX1, and their interacting factors is a promising therapeutic approach to improve survival outcomes of RMS patients.AbstractRhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma that causes significant devastation, with no effective therapy for relapsed disease. The mechanisms behind treatment failures are poorly understood. Our study showed that treatment of RMS cells with vincristine led to an increase in CD133-positive stem-like resistant cells. Single cell RNAseq analysis revealed that MYC and YBX1 were among the top-scoring transcription factors in CD133-high expressing cells. Targeting MYC and YBX1 using CRISPR/Cas9 reduced stem-like characteristics and viability of the vincristine-resistant cells. MYC and YBX1 showed mutual regulation, with MYC binding to the YBX1 promoter and YBX1 binding to MYC mRNA. The MYC inhibitor MYC361i synergized with vincristine to reduce tumor growth and stem-like cells in a zebrafish model of RMS. MYC and YBX expression showed a positive correlation in RMS patients, and high MYC expression correlated with poor survival. Targeting the MYC-YBX1 axis holds promise for improving survival in RMS patients.