Toxicity associated with in vivo administration of adenovirus (Ad) vectors has been linked to activation of both innate and adaptive immune responses. Pre-existing immunity to the prevalent Ad serotypes, acquired by the majority of the human population as a result of natural infections, has the potential to modulate vector efficacy and safety. Previously, we evaluated some aspects of toxicity from systemic Ad vector in vector-naive and pre-immunized rhesus monkeys. In this report, we summarize data from several studies analyzing toxic effects from systemically administered E1/E3-deleted Ad vector in vector-naive and pre-immunized C57BL/6 mice. Our results indicate that pre-immunization can be associated with increased mortality shortly after systemic administration of Ad. Transient leukopenia and thrombocytopenia were observed early post vector infusion in both vector-naive and pre-immunized animals. Pre-exposure to the vector did not prevent induction of pro-inflammatory cytokines; however, pre-immunized mice showed less tissue toxicity. Growth of bone marrow myeloid and erythroid progenitors was transiently inhibited in pre-immunized animals, but only the myeloid progenitors were affected in vector-naive animals. In summary, pre-existing immunity to Ad vector substantially modifies host immune responses to systemic Ad vector.