Selectively targeting the cancer-specific protein-protein interaction (PPI) between Hsp70 and Bim has been discovered as a promising strategy for treating chronic myeloid leukemia (CML). The first Hsp70-Bim PPI inhibitor, S1g-2, has been identified to overcome the on-target toxicity of known Hsp70 inhibitors when it induces apoptosis of CML cells. Herein, we carried out a hit-to-lead optimization of S1g-2, yielding S1g-10, which exhibited a 10-fold increase in Hsp70/Bim suppressing potency. Furthermore, S1g-10 not only exhibited a 5- to 10-fold stronger antitumor activity in the sub-μM range against CML cells than S1g-2 in vitro, but it also overcame BCR-ABL-independent tyrosine kinase inhibitor resistance in CML in vivo depending on the Hsp70-Bim signaling pathway. Moreover, through structure-activity relationship analysis, TROSY-HSQC NMR, molecular dynamics simulation, and point mutation validation, two hydrophobic pockets composed of eight key residues were demonstrated to produce predominant interactions with either Bim or S1g-10, regarded as the "hot-spots" in the Hsp70-Bim PPI interface.