Small molecule inhibitors, such as imatinib, are effective therapies for tyrosine kinase fusions BCR-ABL-TEL-PDGFbetaR-mediated human leukemias, but resistance may develop. The unique fusion junctions of these molecules are attractive candidates for molecularly targeted therapeutic intervention using RNA interference (RNAi), which is mediated by small interfering RNA (siRNA). We developed a retroviral system for stable expression of siRNA directed to the unique fusion junction sequence of TEL-PDGFbetaR in transformed hematopoietic cells. Stable expression of the siRNA resulted in approximately 90% inhibition of TEL-PDGFbetaR expression and its downstream effectors, including PI3K and mammalian target of rapamycin (mTOR). Expression of TEL-PDGFbetaR-specific siRNA (TPsiRNA) significantly attenuated the proliferation of TEL-PDGFbetaR-transformed Ba/F3 cells or disease latency and penetrance in mice induced by intravenous injection of these Ba/F3 cells. Although a 90% reduction in TEL-PDGFbetaR expression was insufficient to induce cell death, stable siRNA expression sensitized transformed cells to the PDGFbetaR inhibitor imatinib or to the mTOR inhibitor rapamycin. TPsiRNA also inhibited an imatinib-resistant TEL-PDGFbetaR mutant, and the inhibition was enhanced by siRNA in combination with PKC412, another PDGFbetaR inhibitor. Although siRNA delivery in vivo is a challenging problem, stable expression of siRNA, which targets oncogenic fusion genes, may potentiate the effects of conventional therapy for hematologic malignancies.