We have previously reported that heat shock protein 70 (HSP70) vaccination induced natural killer (NK) cell activity in patients with chronic myelogenous leukemia (CML). In this study, HSP70 of both autologous and allogeneic origin was found to stimulate IFN-γ production from peripheral blood mononuclear cells of CML patients, as well as of normal subjects. Further investigations demonstrated that the activity of HSP70 was dependent on both NK cells and dendritic cells (DCs). HSP70 did not induce significant IFN-γ production from either NK cells or DCs alone. Mechanistically, we found that HSP70-mediated DC-NK cell crosstalk required cell-cell contact, which could be inhibited completely by neutralizing antibody against NK activating receptor NKG2D. The significance of NKG2D was further corroborated by the finding that HSP70 induced the expression of an NKG2D ligand, the MHC class I chain-related protein A (MICA), on DCs; HSP70-augmented IFN-γ release was abrogated by antibody against MICA. Thus extracellular HSP70, released during either stress or inflammatory cell death, may serve as a critical link between NK and DCs in mounting immune responses against infections, cancers and self-antigens.