Toxoplasma gondii naturally causes a lifelong, asymptomatic infection of the central nervous system (CNS) in immunocompetent humans and rodents. In the CNS, T. gondii establishes a chronic infection by converting to slow-growing bradyzoites that encyst. Consistent with prior studies, we have observed strain-specific differences in encystment, with Pru (type II) parasites encysting faster and in higher numbers than CEP (type III) parasites in pH-stressed fibroblasts, in human and murine neurons, and in vivo. To use these strain-specific differences in the encystment to identify bradyzoite-specific genes, we performed RNAseq on murine neurons infected with Pru or CEP parasites. We identified 502 T. gondii genes that showed more than 2-fold upregulation in Pru compared to CEP. We focused on defining the role of TGME49_207210 (TLO) in encystment because it was one of the top 10 upregulated genes in Pru, had been identified in other studies of bradyzoite genes, had a fitness score of 0, and was predicted to have BFD1 binding sites. We generated a CRISPR-mediated deletion of TGME49_207210 (TLO) in Pru and confirmed that PruΔ207210 had no plaque or growth defect in human foreskin fibroblasts. Conversely, in primary murine neurons, PruΔ207210 had a 50+% reduction in encystment even at 3 days post-infection; complementing the gene back rescued the encystment defect to wild-type levels. In vivo, PruΔ207210 showed no differences in the CNS parasite burden (Q-PCR for B1), cyst size, or cyst numbers at 3 weeks post-infection (wpi) but all three measures were decreased at 8 wpi compared with wild-type Pru. Overall, these data highlight how strain-specific differences can be used to gain insights into chronic infections. Our current work is focused on generating tagged-complemented strains so that we can mechanistically define the cellular and molecular function for this gene.