Experimental autoimmune encephalomyelitis (EAE) induced by active immunization of C57BL/6 mice with peptide from myelin oligodendrocyte protein (MOG_35–55), is a neuroinflammatory, demyelinating disease widely recognized as an animal model of multiple sclerosis (MS). Typically, EAE presents with an ascending course of paralysis, and inflammation that is predominantly localized to the spinal cord. Recent studies have further indicated that inflammation – in both MS and EAE – might initiate within the meninges and propagate from there to the underlying parenchyma. However, the patterns of inflammation within the respective meningeal and parenchymal compartments along the length of the spinal cord, and the progression with which these patterns develop during EAE, have yet to be detailed. Such analysis could hold key to identifying factors critical for spreading, as well as constraining, inflammation along the neuraxis. To address this issue, high-resolution 3-dimensional (3D) confocal microscopy was performed to visualize, in detail, the sequence of leukocyte infiltration at distinct regions of the spinal cord. High quality virtual slide scanning for imaging the entire spinal cord using epifluorescence was further conducted to highlight the directionality and relative degree of inflammation. Meningeal inflammation was found to precede parenchymal inflammation at all levels of the spinal cord, but did not develop equally or simultaneously throughout the subarachnoid space (SAS) of the meninges. Instead, meningeal inflammation was initially most obvious in the caudal SAS, from which it progressed to the immediate underlying parenchyma, paralleling the first signs of clinical disease in the tail and hind limbs. Meningeal inflammation could then be seen to extend in the caudal-to-rostral direction, followed by a similar, but delayed, trajectory of parenchymal inflammation. To additionally determine whether the course of ascending paralysis and leukocyte infiltration during EAE is reflected in differences in inflammatory gene expression by meningeal and parenchymal microvessels along the spinal cord, laser capture microdissection (LCM) coupled with gene expression profiling was performed. Expression profiles varied between these respective vessel populations at both the cervical and caudal levels of the spinal cord during disease progression, and within each vessel population at different levels of the cord at a given time during disease. These results reinforce a significant role for the meninges in the development and propagation of central nervous system inflammation associated with MS and EAE.