Ceratocystis lukuohia is one of two newly described pathogens of Metrosideros polymorpha (‘ōhi‘a) causing Rapid ‘Ōhi‘a Death, a phenomenon that is devastating sections of native forest across the state of Hawai‘i, USA. Ceratocystis lukuohia research has increased as the threat of the pathogen has become more apparent, resulting in a need for efficient production of fungal cultures to meet research demands. Therefore, the temperature and medium-dependent mycelium growth and spore production of three isolates, one from the outbreak area of Leilani Estates and two from beyond the area, were assessed in culture. Fungal growth, measured as mycelium diameter, did not differ for the three isolates after 7 days of incubation. Optimal growth temperatures were 25–30 °C on 10% V8 agar and 25 °C on malt yeast extract agar (MYEA). Spore production, as assessed in one isolate, was most abundant at 25 °C and on MYEA, followed by 10% V8, and MEA. Mycelium growth and spore production on 10% V8 were less variable compared to the other media and, while mean spore production was highest on MYEA, the test isolate produced at least 1 × 10⁶ spores/mL on 10% V8 agar, a commonly used spore concentration for inoculation experiments. Therefore, we recommend growing C. lukuohia at 25 °C on 10% V8 for inoculum production. These results contribute to the growing knowledge base of this newly discovered fungal pathogen and suggest a standardized methodology for propagating C. lukuohia for use in host-pathogen interaction studies.