Cell lineage studies provide an important foundation for experimental analysis in many systems. Drosophila neural precursors (neuroblasts) sequentially generate ganglion mother cells (GMCs), which generate neurons and/or glia, but the birth order, or cell lineage, of each neuroblast is poorly understood. The best-characterized neuroblast is NB7-3, in which GMC-1 makes the EW1 serotonergic interneuron and GW motoneuron; GMC-2 makes the EW2 serotonergic interneuron and a programmed cell death; and GMC-3 gives rise to the EW3 interneuron. However, the end of this lineage has not been determined. Here, we use positively marked genetic clones, bromodeoxyuridine (BrdU) labeling, mutations that affect Notch signaling, and antibody markers to further define the end of the cell lineage of NB7-3. We provide evidence that GMC-3 directly differentiates into EW3 and that the sibling neuroblast undergoes programmed cell death. Our results confirm and extend previous work on the early portion of the NB7-3 lineage (Novotny et al.  Development 129:1027–1036; Lundell et al. [ 2003] Development 130:4109–4121). J. Comp. Neurol. 481:240–251, 2005. © 2004 Wiley-Liss, Inc.