Previous research has emphasized that the basal forebrain partially controls high-order neural processes such as memory. For this reason, it has often been studied in the context of Alzheimer’s and Parkinson’s disease, which both correspond to impaired cognitive function. In spite of the importance of this research, however, there stands much to learn and even revise about our understanding of the neurons which populate the basal forebrain. Neurons expressing the gene T-Box Brain 1 (TBR1) found in the Horizontal Limb Diagonal Band (HDB) and the Substantia Innominata (SI)—both regions within the basal forebrain—have been identified in the past as cholinergic neurons, meaning that they employ acetylcholine as their neurotransmitter. However, immunohistochemical stains of mouse brain sections in the Hevner lab have revealed counterevidence to this claim. We have hypothesized instead that the neurons which populate the HDB and SI are actually glutamatergic neurons, not cholinergic. In order to test this claim, we harvested mouse brains from different ages, ranging from embryonic, postnatal, to adult mice. These brains were embedded in Optimal Cutting Temperature Compound then sectioned these into 12 μm thick slices. These slices were then mounted on slides and stained via immunohistochemical techniques or via endogenous expression. High-magnification images were then collected using AxioVision software to confirm morphology and colocalization of neurons. Employing the above techniques, we have concluded that Tbr1-expressing neurons in the HBD and SI are not cholingeric. Rather, they are glutamatergic neurons. This distinction will be important as research into Alzheimer’s and Parkinson’s diseases develop. An understanding of the precise physical bases for complex, intangible processes is the necessary prerequisite to developing effective therapies.