Glioblastoma is the most common and most lethal primary brain tumor to affect adults. While current treatment options provide temporary recourse, the majority of patients experience tumor recurrence and few survive five years past their initial diagnosis. Recently, tumor microtubes (TMs) were identified in an in vivo model of glioblastoma. These membrane-bound structures formed physical connections between tumor cells, over short and long distances, and facilitated intratumoral communication, invasion, treatment resistance, and post-treatment tumor recovery. To date, this is the first instance of TMs being reported in glioblastoma. The lack of an in vitro model for these structures has delayed further characterization of how TMs form between cells, facilitate intercellular exchange, and how they can be therapeutically targeted to increase treatment susceptibility. The study presented here is the first instance of TMs characterized in an in vitro model of primary glioblastoma (PriGO) cells. These TMs recapitulated many of the structural and functional properties of those observed in vivo, making it a suitable model for further experimentation. Using this model, Rac1, a known orchestrator of cytoskeletal remodeling and motility, was shown to be integral to establishing a TM network between PriGO cells, as demonstrated by siRNA-mediated protein knockdowns. PREX1, a GEF necessary for Rac1 signaling activity, also played a role in PriGO TM formation as evidenced by CRISPR/Cas9-based knockouts. Re-introducing a PREX1 domain with Rac-GEF activity into cells lacking the protein led to a functional rescue of TM growth, thus confirming PREX1’s involvement. Characterizing a cell culture model of glioblastoma TMs is a necessary first step in the study of these structures, ultimately paving the way for future development of therapies that disrupt this network.