Previous studies have shown that transgenic (Tg) mice overexpressing human tau protein develop filamentous tau aggregates in the CNS. The most abundant tau aggregates are found in spinal cord and brainstem in which they colocalize with neurofilaments (NFs) as spheroids in axons. To elucidate the role of NF subunit proteins in tau aggregate formation and to test the hypothesis that NFs are pathological chaperones in the formation of intraneuronal tau inclusions, we crossbred previously described tau (T44) Tg mice overexpressing the smallest human tau isoform with knock-out mice devoid of NFL (NFL-/-) or NFH (NFH-/-). Depletion of NF subunit proteins from the T44 mice (i.e., T44;NFL-/- and T44;NFH-/-), in particular NFL, resulted in a dramatic decrease in the total number of tau-positive spheroids in spinal cord and brainstem. Concomitant with the reduction in spheroid number, the bigenic mice showed delayed accumulation of insoluble tau protein in the CNS, increased viability, reduced weight loss, and improved behavioral phenotype when compared with the single T44 Tg mice. These results imply that NFs are pathological chaperones in the development of tau spheroids and suggest a role for NFs in the pathogenesis of neurofibrillary tau lesions in neurodegenerative disorders that contain both NFs and tau proteins.