Toxoplasmosis, caused by Toxoplasma gondii, is a major global health concern due to its high prevalence, zoonotic transmission, and economic impact on livestock. The parasite’s life cycle includes asexual, pre-sexual, and sexual stages, the latter responsible for oocyst shedding and genetic recombination. Understanding sexual differentiation is critical, but access has been limited because these stages occur only in the feline intestinal epithelium. Recent studies identify host metabolic cues and the microrchidia (MORC) protein complex as regulators of sexual commitment. We optimize in vitro approaches to enrich pre-sexual and sexual stages by combining a human retinal epithelial cell line and murine intestinal organoids with FELIX medium, which mimics feline intestinal biochemistry, and conditional MORC depletion. This system increases stage-specific gene expression and marker detection, demonstrating synergistic effects of host environment and genetic regulation. Our findings provide accessible models to study T. gondii sexual differentiation, with implications for controlling transmission and genetic diversity.