During initial infection, Toxoplasma gondii differentiates into tachyzoites that rapidly disseminate throughout the host, invading nucleated cells. A proportion of tachyzoites differentiate into slow-growing bradyzoites, which form cysts that are mainly localized within the brain and muscle tissues, but the molecular basis of this differentiation was unknown. Now, Waldman et al. identify a master regulator of chronic-stage differentiation in T. gondii. The authors used Cas9-mediated genetic screening and single-cell transcriptional profiling to find putative regulators of differentiation and identified the transcription factor BFD1. ΔBFD1 parasites were unable to differentiate in cell culture or form brain cysts in mice, and BFD1 expression was sufficient to induce differentiation in cell culture. BFD1 was found to bind to the promoters of numerous differentially regulated stage-specific genes, suggesting that BDF1 directly activates differentiation.