Extensive antibiotic use in the poultry industry has given rise to concerns of the proliferation and potential zoonotic transmission of antimicrobial-resistant pathogens. To mitigate the extent of antibiotic dosing, probiotics have been proposed as an alternate means of enhancing chick immune function by boosting host defense peptide (HDP) production. This study measured chick immune response to in ovo supplementation of a commercial poultry probiotic when subsequently exposed to a mimicked viral threat using polyinosinic-polycytidylic acid (poly(I:C)). To assess systemic immune responses to these conditions, real-time PCR was conducted to measure changes in expression of five immune-related genes in the spleen: TLR-3, AvBD-2, IFN-α, IFN-β, and IL-6. The chicks that received only the probiotic treatment saw a downregulation of TLR-3 (p=0.0013) and an upregulation of AvBD-2 (p=0.0105). The expression of IL-6 tended towards downregulation (p=0.06) with a potential interaction between the probiotic and poly(I:C) treatments (p=0.0794). Upregulation of AvBD-2 indicates a higher basal output of the chick's innate immune system upon early exposure to probiotics. These results suggest that enhanced systemic immune response in chicks can be achieved through early application of probiotics and is occurring through immunomodulation pathways that have not been fully elucidated.