In order to gain global pathway perspective of ex vivo viral infection models using human peripheral blood mononuclear cells (PBMCs), we conducted expression analysis on PBMCs of healthy donors. RNA samples were collected at 3 hours and 24 hours after PBMCs were challenged with the TLR3 agonist poly(I:C), and analyzed by internally developed cDNA microarrays and TaqMan PCR. Our results demonstrate that poly(I:C) challenge is able to elicit certain gene expression changes similar to acute viral infection. Hierarchical cluster revealed distinct pattern of immediate early, early-to-late, and late gene regulation patterns. The early responses were innate immune responses that involve TLR3, the NFB dependent pathway and the IFN-stimulated pathway, whereas the late responses were mostly cell mediated immune response that involves activation of cell adhesion, cell mobility and phagocytosis. Overall, our results expanded the utilities of this ex vivo model, which could be used to screen molecules that can modulate viral stress induced inflammation, in particular those mediated via Toll-like receptors.