ABSTRACT Animals build their multicellular bodies with diverse types of cells that perform and integrate distinct functions. Animals, however, are not unique in their capacity to generate distinct cell types. In fact, their closest living relatives, a group of aquatic, unicellular, bacterivorous protists called choanoflagellates, detect biotic and abiotic cues to differentiate into phenotypically and functionally distinct cell types in different environments. Choanoflagellates also possess critical genes that regulate animal cell differentiation during development, supporting the hypothesis that cell differentiation mechanisms evolved prior to the origin of animals and became integral in animal and choanoflagellate biology. Although nearly 800 million years of animal evolution has shaped human biology since we last shared a common ancestor with choanoflagellates, the commonalities in genetic toolkits and cytological characteristics indicate that choanoflagellates have tremendous potential as microeukaryotic models to investigate the core functions of genes that regulate cell differentiation. During my postdoc, I pioneered the first methods for gene delivery and genome editing in the choanoflagellate Salpingoeca rosetta to realize its full potential as a model system. My lab continues to propel those methods for the discovery of the molecular mechanisms that drive environmentally-triggered cell differentiation in S. rosetta. This proposal supports our research mission by using the molecular tools I developed to dissect putative regulatory pathways that emerge from functional genomic surveys. In particular, we focus on homologs of RNA-binding proteins that form the animal germline and and/or maintain pluripotency. Moreover, we strive to develop our nascent genetic tools into scalable, easy methods that enable genome-wide screens of cell differentiation regulators. Overall, this work will contribute a new, functional comparison to illuminate the origin and evolution of cell differentiation pathways in choanoflagellates and animals, which I anticipate will uncover core functions of biomedically important genes that originated before choanoflagellates and animals diverged.