Viruses are the most successful genetic entity on earth, consisting of only a viral genome, a protein capsid, and an optional lipid outer layer. Viruses primarily use virions, or progeny viral particles, to disseminate their genetic information between infected host cells. While the genomic packaging and virion assembly strategies of many viruses are well understood, the processes used by several important human viruses remain unknown. Despite its importance as a model nuclear-replicating DNA virus and its widespread use as a gene delivery vector, the mechanism of adenovirus (AdV) genome packaging and virion assembly is still debated. Achieving a greater understanding of AdV genome packaging and virion assembly will provide further insight into fundamental processes used by viruses and cells to organize and compartmentalize complex biological processes in a crowded cellular environment. Additionally, it will facilitate improvements in the development and production of recombinant AdV vectors for therapeutic purposes. Thus, this thesis aims to address two key questions regarding AdV genome packaging and capsid assembly using infection of human cells with human AdV: 1) How does the AdV L1-52/55 Kilodalton (52K) protein coordinate and regulate AdV genome packaging and capsid assembly during the late stage of infection? 2) How is the function of the 52K protein regulated and maintained during infection? First, we use multiple in vitro, biochemical, and genetic approaches to investigate the function of the AdV 52K protein during infection. We show that 52K forms viral biomolecular condensates (BMCs) in infected cell nuclei that organize AdV capsid proteins and interact with AdV genomes. Furthermore, we demonstrate a requirement of these 52K BMCs for AdV infectious progeny production. Here, we reveal a novel function of the AdV 52K protein and provide support for the concurrent model of AdV genome packaging and capsid assembly. Second, we investigate the regulation of 52K function during AdV infectious progeny production using a combination of proteomics and molecular biology. We identify several 52K phosphorylation sites that regulate the material properties of 52K BMCs and serve to maintain their dynamic, liquid-like properties throughout AdV infection. Moreover, we demonstrate that 52K phosphorylation serves to promote AdV infectious progeny production. Here, we uncover a regulatory mechanism used by AdV to ensure continued functionality of 52K BMCs and enable robust infectious progeny production. Collectively, our studies shed light on the complex mechanism of AdV genome packaging and progeny production with wider implications for virology, cell biology, and the production of recombinant AdV vectors.