This work has focused on neurotransmitter specification in the developing fruit fly brain. Neuroblasts constitute the fundamental developmental units of the insect nervous system, each generating a unique lineage. Our understanding of neuroblast lineages at the anatomical level contrasts sharply with a lack of understanding of their neurotransmitter phenotypes, overlooking a critical attribute of individual neurons. This has been a conspicuous gap in our understanding in this otherwise powerful and influential model system. For the vast majority of embryonic lineages it is unknown which of the three main neurotransmitters (GABA, Acetylcholine, or Glutamate) their progeny produces. During my PhD, I have developed methods that make it possible to identify neurotransmitter type with confidence in the developing brain via a combination of genetic reporters and immunohistochemistry. I have used these tools to try and characterise neurotransmitter patterning in the first instar larval ventral nerve cord, and found indications that there is a higher ratio of inhibitory : excitatory neurons than might be expected. I also used these tools to characterise the neurotransmitter phenotype of various specific neuroblast lineages, particularly the lineage of neuroblasts 5-3 and 3-5. Through my characterization of neurotransmitter phenotypes, I discovered that there are many cells in the ventral nerve cord that are not labelled with reporters for the key neurotransmitters – GABA, acetylcholine, glutamate, and neuropeptides. In trying to explain this phenomenon, I discovered that there is widespread expression of the Drosophila Glycine-Transporter throughout the larval ventral nerve cord and brain, which previously had only been shown in the adult brain. I then found that down-regulation of Glycine-Transporter expression is lethal during second or third instar and leads to a curious phenotype of larvae being partially or fully tilted so they are dorsal side-up. I also explored the use of live imaging in Drosophila embryos to investigate the role of birth order in neurotransmitter specification, although technical constraints prevented firm conclusions from being reached. Finally, I attempted to manipulate neurotransmitter specification via apoptosis mutants and misexpression of clock-gene Hunchback during embryogenesis, although the phenotypes produced were so severe that analysis was difficult and no firm conclusions could be reached.