Programmed cell death-1 (PD-1) is responsible for T cell exhaustion during chronic viral infections and is expressed on a variety of immune cells following activation. Despite its importance, the mechanisms that regulate PD-1 in cell types other than CD8 T cells are poorly defined. In this study, the molecular mechanisms for inducing PD-1 expression in CD4 T cells, macrophages, and B cells were explored. In CD4 T cells, PD-1 induction following TCR stimulation required NFAT, as the calcineurin/NFAT pathway inhibitor cyclosporin A was able to block PD-1 induction in a manner similar to that seen in CD8 T cells. In contrast, LPS but not PMA and ionomycin stimulation was able to induce PD-1 expression in macrophages in a manner insensitive to cyclosporin A–mediated inhibition. B cells could use both pathways, although the levels of PD-1 expression were highest with PMA and ionomycin. An NF-κB binding site located upstream of the gene in conserved region C was required for NF-κB–dependent PD-1 gene activation in macrophages. Chromatin immunoprecipitation showed NF-κB p65 binding to this region following stimulation of macrophages with LPS. PD-1 induction was associated with histone modifications characteristic of accessible chromatin; however, in contrast to CD8 T cells, conserved region B in macrophages did not lose CpG methylation upon stimulation and PD-1 expression. The linkage of TLR/NF-κB signaling to the induction of PD-1 suggests the possibility of an opportunistic advantage to microbial infections in manipulating immune inhibitory responses.