Toxoplasmosis is an infectious disease caused by the intracellular, obligate Apicomplexan parasite Toxoplasma gondii. Approximately one-third of the global human population is chronically infected with Toxoplasma, and no treatment exists to clear the latent reservoir, putting immunosuppressed patients at risk of reactivation and severe disease. Only a handful of genes have been identified to be important for Toxoplasma differentiation from the acute ‘tachyzoite’ form into the slow-growing latent ‘bradyzoite’ form, including a master transcription factor – BFD1 which is necessary and sufficient for this process. To expand our understanding of the underlying molecular pathway controlling Toxoplasma differentiation, our lab recently performed a series of in vitro and in vivo CRISPR screens, identifying over 30 new genes, including a series of nucleic acid-binding proteins (NBPs) and an E3 ubiquitin ligase complex (GID). Focussing on three NBPs, we show that two are localised in the parasite’s cytoplasm, whilst the other is found in the nucleus. Using quantitative imaging and sophisticated genetics we show that two NBPs likely operate early in the differentiation program upstream of BFD1, whilst the other likely more constitutively required. We show that the level of these proteins is regulated by the E3 ubiquitin ligase. We are currently determining how these NBPs act to control translation repression during bradyzoite development. This study therefore shows the importance of RNA regulation in controlling fate determination in this single celled eukaryote and their role in pathogenesis.