To understand how vaccines initiate adaptive immune responses, it is necessary to study how they interact with APCs such as dendritic cells (DCs). In this study, we analyzed interactions between recombinant adenovirus (Ad) vectors and mouse DCs. Mouse bone marrow-derived DCs transduced with Ad vectors produced type I IFN, which promoted the maturation of both transduced and bystander DCs. DCs transduced with a vector derived from a chimpanzee Ad serotype (AdC68) produced more type I IFN and matured more efficiently compared with DCs transduced with a vector derived from a human Ad serotype (AdHu5). Both vectors stimulated type I IFN production independently of viral transcription, replication, and TLR signaling. However, each vector induced type I IFN through distinct pathways; whereas AdHu5 vectors required phosphoinositide-3-OH kinase for type I IFN induction, AdC68 vectors did not. Both vectors induced strong transgene product-specific CD8+ T cell responses in wild-type mice. DCs isolated from mice that have a defect in type I IFN signaling failed to undergo full maturation after Ad vaccination, but surprisingly, these mice mounted strong transgene product-specific CD8+ T cell responses. In these mice, we were able to detect a small number of transduced DCs that expressed high levels of costimulatory molecules, and these DCs were able to stimulate transgene product-specific CD8+ T cells. Thus, type I IFN signaling is an important component of Ad-mediated DC maturation but is dispensable during the generation of transgene product-specific CD8+ T cell responses.