Natural killer (NK) cells hold promise for improving the therapeutic potential of allogeneic hematopoietic transplantation, but their effectiveness is limited by inhibitory HLA types. We sought to overcome this intrinsic resistance by transducing CD56+CD3- NK cells with chimeric receptors directed against CD19, a molecule widely expressed by malignant B cells. An abundance of NK cells for transduction was secured by culturing peripheral blood mononuclear cells with K562 cells expressing the NK-stimulatory molecules 4-1BB ligand and interleukin 15, which yielded a median greater than 1000-fold expansion of CD56+CD3- cells at 3 weeks of culture, without T-lymphocyte expansion. Expression of anti-CD19 receptors linked to CD3zeta overcame NK resistance and markedly enhanced NK-cell-mediated killing of leukemic cells. This result was significantly improved by adding the 4-1BB costimulatory molecule to the chimeric anti-CD19-CD3zeta receptor; the cytotoxicity produced by NK cells expressing this construct uniformly exceeded that of NK cells whose signaling receptors lacked 4-1BB, even when natural cytotoxicity was apparent. Addition of 4-1BB was also associated with increased cell activation and production of interferon gamma and granulocyte-macrophage colony-stimulating factor. Our findings indicate that enforced expression of signaling receptors by NK cells might circumvent inhibitory signals, providing a novel means to enhance the effectiveness of allogeneic stem cell transplantation.