<p>Stroke triggers a rapid and complex immune response that is not yet fully understood, especially within hours after an ischemic infarct. Our previous study in stroke patients revealed a significant increase in interferon-gamma (IFN-γ) immediately (hyperacute) and downstream of the ischemic ictus, within the arterial compartment. The present study investigated the source, inciting factors, and role of IFN-γ in a preclinical murine model. Stroke was produced using transient middle cerebral artery occlusion, and immune cells within the arterial vasculature distal to the occlusion (pre- and post-occlusion) were characterized using flow cytometry. Compared with the control samples, the post-occlusion samples presented an increase in IFN-γ⁺ and CD69⁺ cells, whereas no significant increase was detected in IL17⁺, IL4⁺, and CD25⁺ cells. Further analysis of the IFN-γ⁺ population revealed two novel attributes. First, interrogation of the identity of these IFN-γ⁺ cells revealed that the increase in IFN-γ production was largely driven by CD14⁺ myeloid cells in the post-occlusion sample, with negligible contributions from other canonical IFN-γ-producing cells (CD4, CD8, NK). Second, the IFN-γ⁺ cells exhibited two distinct clusters, an IFN-γ^low and an IFN-γ^hi population. Further analysis revealed that the IFN-γ ^low population was largely composed of CD14⁺ myeloid cells, whereas the IFN-γ^hi population was dominated by CD4⁺ T-cells. To explore the conditions driving IFN-γ production, an in vitro ischemia model involving oxygen-glucose deprivation (OGD) was employed. Co-culturing of naïve splenocytes with OGD-treated CNS cells and OGD-derived supernatant resulted in a significant increase in IFN-γ⁺CD14⁺ cells, as compared to normoxic controls, an effect that coincided with marked loss of DAPI⁺ and NeuN⁺DAPI⁺ cells in mixed cortical (neuronal and glial) cultures. In summary, this study identified intra-arterial CD14⁺ myeloid cells as novel early sources of IFN-γ in the hyperacute phase of stroke, a role traditionally attributed to adaptive immune cells. Using in vivo and in vitro ischemia models, the findings reveal that injury-associated signals from CNS cells are sufficient to directly induce IFN-γ production in CD14⁺ myeloid cells, redefining early stroke immunopathology and uncovering a potential target for timely immunomodulation.</p>