Despite growing evidence for platelets as active players in infection and immunity, it remains unresolved whether platelets contribute to, or are key elements in the development of neuroinflammation. Using the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, we identified platelet accumulation in the circulation by 7-day postinduction (dpi), ahead of clinical onset which occurs at 13-14 dpi. By inducing platelet depletion between 7 and 16 dpi, we demonstrate an association between platelet accumulation in the spinal cord and disease development. Additionally, we provide evidence for platelet infiltration in the white and gray matter parenchyma, but with different outcomes. Thus, while in white matter platelets are clearly associated with lesions, in gray matter large-scale platelet infiltration and expression of the platelet-specific molecule PF4 are detectable prior to T cell entry. In the retina, platelet accumulation also precedes clinical onset and is associated with significant increase in retinal thickness in experimental relative to control animals. Platelet accumulation increases over the disease course in this tissue, but without subsequent T cell infiltration. These findings provide definitive confirmation that platelet accumulation is key to EAE pathophysiology. Furthermore, they suggest an undescribed and, most importantly, therapeutically targetable mechanism of neuronal damage.