Technical Abstract: Few experimental models are available for studying a diverse assemblage of tissue cyst-forming coccidian parasites, including members of the genus Besnoitia. Here, we describe how suppression of cellular immunity rendered outbread mice susceptible to two isolates of Besnoitia darlingi originating in naturally infected opussums, and the subsequent development of long-term tissue cultures that maintained viable parasites. Numerous cysts typical of parasites belonging to the Apicomplexan genus Besnoitia were found in lungs, liver, kidneys, spleen, heart, tongue, diaphragm, abdominal muscles, skin, and ears of the opossums. The light microscopic and ultrastructural characteristics of the cysts and bradyzoites were consistent with that of Besnoitia darlingi, and this diagnosis was also supported based on comparative sequence analysis at each of two genetic loci. Bradyzoites recovered from crushed cysts from the infected opossums were inoculated into outbred Webster laboratory mice by intraperitoneal and subcutaneous routes. The tachyzoites induced severe systemic infection in mice treated with dexamethasone, but were not pathogenic to mice treated with sulfadiazine. Isolates recovered from the inoculated mice were viable and could be maintained in long-term tissue cultures.