Objective: This study investigated two approaches, short hairpin RNA (shRNA) and the potent ABL inhibitor, dasatinib, alone and together, to achieve complete inhibition of BCR-ABL activity in Philadelphia-positive (Ph+) cells. Materials and Methods: shRNA specific for BCR-ABL b3a2 were delivered, by lentiviral transduction or electroporation, to K562 cells, with or without dasatinib. mRNA and protein knockdown were measured by quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and Western blotting. BCR-ABL activity was assessed by intracellular flow cytometry for pCrkL. Cell death and apoptosis were assayed using trypan blue exclusion, Annexin-V, and active caspase-3 staining. Results: Forty-eight hours after transduction or electroporation of shRNA, BCR-ABL mRNA, and protein were reduced by 75% and >90%, respectively, and sustained for 5 days. Lentiviral delivery and electroporation were equally effective. pCrkL was inhibited in association with cell death. By 5 days after transduction or electroporation, viable cells represented 50% of input, with a 12-fold reduction vs control, which expanded 6-fold. When shRNA, titrated by green fluorescent protein into low and high, was combined with dasatinib (concentration range, 0-10 nM), low shRNA was additive with low dasatinib (0.6 and 1 nM), leading to inhibition of pCrkL, induction of activated caspase-3, expression of Annexin-V, and marked reduction in viable cells. Conclusion: These results confirm that by lowering BCR-ABL levels with shRNA, complete inhibition of oncoprotein activity can be achieved with a lower concentration of dasatinib, thus providing a rationale for combining these approaches in the setting of high target expression, such as found in advanced phase disease and in the stem cell compartment. © 2009 ISEH - Society for Hematology and Stem Cells.