The midbrain is a critical region of the brainstem that houses the cranial nuclei necessary for coordinating eye movements. It has been observed that neurons in the midbrain express T-box Brain 2 (Eomes/Tbr2), a transcription factor generally associated with development of glutamatergic neurons. Because it has such a diverse role throughout the development of multiple neural structures (i.e. cerebral cortex, olfactory bulb, retina, and cerebellum), Tbr2 is thought to be implicated in the development of certain forms of autism. The purpose of our experiments was thus two-fold: first, we investigated the neurotransmitter phenotype of Tbr2+ neurons in the midbrain. Second, we wanted to identify the function of Tbr2+ neurons in the brain by studying these populations in relation to known, molecular markers of specific, anatomical features. In order to accomplish these aims, we collected tissues at various ages from transgenic mice. We utilized endogenous reporters such as VGlut2-CreAi14 and Tbr2mTnG alongside immunohistochemical staining with the following antibodies: urocortin-1 (UCN-1; which marks the centrally-projecting Edinger-Westphal nucleus, EWcp, in the postnatal brain); cocaine- and amphetamine-regulated transcript (CART, which marks EWcp in the embryonic and postnatal brain); and 2H3 (which marks neurofilaments). We then used a confocal microscope to visualize fluorescence and collect images. We found that Tbr2+ neurons in the midbrain were glutamatergic but not cholinergic. We also found that Tbr2 neurons were not Edinger-Westphal cells, leaving their ultimate anatomical identity unknown. Understanding the identity of Tbr2 cells is a prerequisite for beginning to understand the constellation of features that make up autistic spectral disorder, thereby guiding future clinical and scientific research.