Influenza A virus (IAV) consists of eight viral RNA (vRNA) segments that are replicated in the host cell nucleus and transported to the plasma membrane for packaging into progeny virions. We have previously proposed a model where sub-complexes of vRNA export from the nucleus and assemble en route to the plasma membrane. However, the role of host cytoskeletal proteins in the cytoplasmic assembly of IAV vRNA segments remains unknown. Previous studies have suggested that IAV vRNA segments transport via Rab11A-containing recycling endosomes (RE) and use both microtubules (MT) and actin. Rab11A-RE primarily transport along MT; therefore, investigation into the role of MT on vRNA assembly is warranted. We explored the role of MT in vRNA assembly and replication using multiple IAV strains in various cell types, including primary human airway epithelial cells. We observed that Rab11A localization was altered in the presence of MT-depolymerizing drugs, but growth of IAV in all cell types was unchanged. Fluorescent in situ hybridization was performed to determine the role of MT in the assembly of multiple vRNA segments. Unexpectedly, we found that vRNA-vRNA association in cytoplasmic foci was independent of MT. Given the disparity of localization between Rab11A and vRNA segments in the absence of intact MT filaments, we analyzed the three-dimensional spatial relationship between Rab11A and vRNA in the cytoplasm of infected cells. We found that Rab11A and vRNA colocalization is dependent upon dynamic MT filaments. Taken together, our data suggest that cytoplasmic transport of influenza vRNA may include a Rab11A-RE independent mechanism.Importance IAV infections cause large public health burden through seasonal epidemics and sporadic pandemics. Pandemic IAV emerge through reassortment of vRNA in animal or human hosts. Elucidating the mechanism of intracellular dynamics of IAV assembly is necessary to understand reassortment. Our results describing the role of MT in vRNA transport and assembly extend previous studies characterizing vRNA assembly. This study is the first to assess the role of MT on influenza replication in human bronchial airway epithelial cells. In addition, we present novel data on the role of MT to facilitate association between distinct vRNA segments. Interestingly, our results suggest that progressive assembly of vRNA segments may be cell type dependent and that vRNA may transport through the cytoplasm without Rab11A-RE in the absence of intact MT. These results enhance our current understanding of vRNA assembly and the role of cytoskeletal proteins in that process.