The gastrointestinal (GI) microbiome plays an important role in the development and function not only of the GI, but also the immune system. Products such as probiotics represent promising treatments for improving animal health through the GI, but their modes of action are largely still unknown. Previous research in our laboratory has demonstrated supplementation with a lactic acid bacteria probiotic product alters host energy partitioning in immune tissues, including increased ATP production and consumption in circulating leukocytes; and was associated with a more rapid antibody response to antigen. To better understand the communication between probiotics and the immune system our laboratory has focused on characterizing how supplementation affects the microbiome and host systems. We hypothesized that the changes previously observed in immune tissue activity and energy metabolism were regulated by a probiotic‐stimulated factor in the serum. We examined the ability of serum isolated from probiotic‐fed animals to augment ATP production in vitro. Chicken lymphocyte cell lines were cultured for 4 days in media supplemented with serum from treated and control birds. Cells cultured in serum from probiotic‐fed birds had higher levels of ATP (P < 0.05) compared to controls. Transcriptomic analysis of these cells suggest an increase in genes associated with cell survival and differentiation, and signaling via TGF‐β, IFN‐γ, IL‐7, and IL‐1β. Analysis of the metabolites in serum and digesta from these animals identified several metabolite changes correlate with the change in ATP levels in lymphocytes and putative changes in pro‐ and anti‐inflammatory cytokine production.