The 190kD (p190) and 210kD (p210) Bcr-Abl proteins are responsible for the pathophysiology of Philadelphia chromosome (Ph)+ leukemia. We applied RNA interference (RNAi) to specific killing of p190+ cells, and determined the optimal sequences for gene silencing in the BCR, junctional and ABL regions of p190, respectively. Then, p190+ and p210+ cells were infected with lentiviral vectors encoding these shRNAs, resulting in efficient killing of p190+ cells, while p210+ cells were only sensitive to shBCR and shABL. In p190-transformed Ba/F3 cells, silencing of p190 specifically inhibited tyrosine phospohorylation of Stat5 prior to their death, but did not affect phosphorylation of Jak2, Akt or MEK1/2. In contrast, downregulation of p190 by their treatment with 17-allylamino-17-demetoxygeldanamycin (17-AAG) was associated with reduced protein levels of Jak2, Akt and MEK1/2. shRNA targeting p190 collaborated additively with imatinib and 17-AAG in growth inhibition of Ba/F3-p190wt and imatinib-resistant Ba/F3-p190Y253H cells. Collectively, RNAi-mediated silencing of p190 is a promising option both for delineating signal transduction and for therapeutic application in 190+ leukemia.