Author Summary Toxoplasma gondii, an important human pathogen, is an obligate intracellular parasite, thus getting in and out of cells is key for its survival. The process by which Toxoplasma exits cells, known as egress, is controlled by calcium fluxes and can be triggered by ionophores. In vivo, rapid egress from the host cell has been identified as a means to escape attack by the innate immune system. At the molecular level, calcium dependent events in this parasite are regulated in part by plant like calcium dependent kinases, which share no homology to human kinases and are thus ideal drug targets. In this study we revisited 4 mutant parasite lines that were independently selected for an inability to undergo egress in response to ionophores. In all four mutants we have identified the Calcium Dependent Kinase 3 as the gene responsible for the defects. We have shown that two of these mutants, which are in a genetic background that allows virulence studies, also have a strong phenotype in vivo. That is, the parasites fail to form latent stages in mice. This work provides important information that a single kinase is responsible for the formation of latent stages that are important for transmission of the parasite.