Abstract Of interest to the etiology of demyelinating autoimmune disease is the potential to aberrantly activate CD4+ T cells due to cross-recognition of multiple self-epitopes such as has been suggested for myelin oligodendrocyte glycoprotein epitope 35–55 (MOG35–55) and neurofilament medium protein epitope 15–35 (NFM15–35). NFM15–35 is immunogenic in C57BL/6 mice but fails to induce demyelinating disease by polyclonal T cells despite having the same TCR contact residues as MOG35–55, a known encephalitogenic Ag. Despite reported cross-reactivity with MOG-specific T cells, the polyclonal response to NFM15–35 did not expand threshold numbers of MOG38–49 tetramer–positive T cells. Furthermore, NFM lacked functional synergy with MOG to promote experimental autoimmune encephalomyelitis because NFM-deficient synonymous with knockout mice developed an identical disease course to wild-type mice after challenge with MOG35–55. Single-cell analysis of encephalitogenic T cells using the peptide:MHC monomer-based two-dimensional micropipette adhesion frequency assay confirmed that NFM was not a critical Ag driving demyelinating disease because NFM18–30–specific T cells in the CNS were predominantly reactive to MOG38–49. The absence of NFM contribution to disease allowed mapping of the amino acids required for encephalitogenicity and expansion of high-affinity, MOG-specific T cells that defined the polyclonal response. Alterations of N-terminal residues outside of the NFM15–35 core nonamer promoted expansion of high-affinity, MOG38–49 tetramer–positive T cells and promoted consistent experimental autoimmune encephalomyelitis induction, unlike mice challenged with NFM15–35. Although NFM15–35 is immunogenic and cross-reactive with MOG at the polyclonal level, it fails to expand a threshold level of encephalitogenic, high-affinity MOG-specific T cells.