Antecedent therapy for ischemic brain injury has the potential to protect a large, high-risk patient population from the devastating effects of cerebral ischemia associated with cardiac surgery. Substantial evidence has shown that preconditioning with a modestly damaging stimulus induces powerful endogenous neuroprotection. Pharmacological agents that stimulate toll-like receptors (TLRs) induce robust neuroprotective effects as preconditioning stimuli against cerebral ischemia in mouse and nonhuman primate models of stroke. Here we describe the progress of our preclinical development of TLR agonists as antecedent therapy against cerebral ischemic injury. The objective was to discuss studies that begin with in vitro validation in cell cultures to in vivo efficacy studies using mouse and nonhuman primate models of stroke, with particular emphasis on the TLR9 agonist CpG oligonucleotide. We provide an in-depth discussion of our novel rhesus macaque stroke model and cover the progress we have made in therapeutic testing and evaluation in these animals. These studies represent a logical path for the development of TLR agonists as antecedent therapy for the prevention of the damaging neurological complications resulting from cerebral ischemic injury.