Understanding the determinants of the host innate immune response to systemic administration of adenoviral (Ad) vectors is critical for clinical gene therapy. Acute toxicity occurs within minutes to hours after vector administration and is characterized by activation of innate immune responses. Our data indicate that in mice, indicators of vector toxicity include elevations of cytokine levels, liver transaminase levels and thrombocytopenia. To discern potential targets for blunting this host response, we evaluated genetic factors in the host response to systemically administered first-generation Ad vectors (FGV) and helper-dependent Ad vectors (HDV) containing beta-galactosidase expression cassettes. A preliminary screen for modulation of vector-induced thrombocytopenia revealed no role for interferon-gamma, mast cells or perforin. However, vector-induced thrombocytopenia and interleukin 6 (IL-6) expression are less evident in tumor necrosis factor alpha (TNFalpha)-deficient mice. Moreover, we also demonstrated that TNFalpha blockade via antibody or huTNFR:Fc pretreatment attenuates both thrombocytopenia (>40% increase in platelet count) and IL-6 expression (>80% reduction) without affecting interleukin 12 , liver enzymes, hematological indices or vector transduction in a murine model. Our data indicate that the use of HDV, in combination with clinically approved TNFalpha immunomodulation, may represent an approach for improving the therapeutic index of Ad gene therapy for human clinical trials.