SYK tyrosine kinase has emerged as a master regulator of cellular resistance to oxidative stress (OS) by mediating the activation of the anti-apoptotic nuclear factor kappaB and phosphatidylinositol-3 kinase/AKT pathways after OS exposure. Here, we present unprecedented experimental evidence that polo-like kinase 1 (PLK1) is the upstream regulator of SYK in B-lineage acute lymphoblastic leukaemia (ALL) cells. Selective inhibition of PLK-1 with the leflunomide metabolite analogue alpha-cyano-beta-hydroxy-beta-methyl-N-[4-(trifluoromethoxy) phenyl]-propenamide/LFM-A12 abolished the resistance of B-lineage ALL cells to OS by preventing the activation of the anti-apoptotic SYK signal transduction pathway. Notably, LFM-A12 treatments at non-cytotoxic concentrations resulted in marked augmentation of clonogenic death in resistant human B-lineage ALL cell lines challenged with OS. Further, LFM-A12 augmented OS-induced apoptosis of chemotherapy-resistant primary leukaemic cells from relapsed B-lineage ALL patients in vitro and markedly potentiated the in vivo anti-leukaemic activity of total body irradiation (TBI) against leukaemia-initiating cells in severe combined immunodeficient mouse xenograft models of B-lineage ALL. This study is the first to identify PLK1 as a regulator of SYK tyrosine kinase and a molecular target to overcome SYK-mediated resistance of B-lineage ALL cells to OS.