The immune system is a multitiered network that at the first level uses changes to intracellular signaling proteins to commit cells to determined fates. At the second tier, cells interact with one another via specifically expressed surface receptors and their cognate signaling molecules. At the third level, the local environments of immune cells change the outcomes of intracellular signaling pathways and thereby the role of cells during immune challenge. The interplay among these three tiers allows the distinct cell types of the immune system to respond cohesively to eliminate foreign Ags. In this study, using phosphospecific flow cytometry, we analyze elements of these network tiers by generating profiles of single-cell phosphoprotein responses in B cells, T cells, and myeloid cells to a number of mechanistically and clinically relevant cytokines (IFN-gamma, GM-CSF, IL-2, and IL-10) as well as LPS at key regulatory interfaces (Jak-Stat and MAPK pathways). The stimuli typically induced phosphorylation of specific signaling pathways and exerted their effects on distinct subsets of immune cells. However, upon comparison of stimulation in vitro and in vivo, we noted that signaling pathway specificity and cell type specificity were influenced strongly by the external environment. When taken from the in vivo environment, certain cell subsets became hypo- or hyper-responsive, showed profound differences in sensitivity to cytokine levels, or displayed altered phosphorylation kinetics. Thus, simultaneous analysis of the three tiers of the immune system network illustrates the principles by which immune regulation is context dependent and how in vitro culture systems compare with the in vivo environment.