Engaging the immune system to attack neoplastic glial cells in the brain may be a promising approach to eliminate glioblastoma (GBM), a deadly form of primary brain cancer with low median survival. A bispecific fusion protein, anti-CD3/chlorotoxin (ACDClx), has been developed to engage cytotoxic T cells for destruction against GBM with little to no expected toxicity to surrounding healthy tissue. Previously, ACDClx has been demonstrated to induce calcium flux in T cells, indicating activation when cultured with GBM cells in vitro. Here, ACDClx fails to demonstrate successful binding to the CD3 domain of the T-cell receptor on CD4 T cells in vitro and fails to bind GBM cells despite demonstrated binding of chlorotoxin to the same cell line. This data warrants further investigation into the binding characteristics of ACDClx to target cells.