Abstract: The developmental cycle of Myxococcus xanthus involves the coordination of many hundreds of thousands of cells aggregating to form mounds known as fruiting bodies. This aggregation process begins with the sequential formation of more and more cell layers. Using three-dimensional confocal imaging we study this layer formation process by observing the formation of holes and second layers within a base monolayer of M xanthus cells. We find that cells align with each other over the majority of the monolayer forming an active nematic liquid crystal with defect point where cell alignment is undefined. We find that new layers and holes form at positive and negative topological defects respectively. We model the cell layer using hydrodynamic modeling and find that this layer and hole formation process is driven by active nematic forces through cell motility and anisotropic substrate friction.