Methods are described for the in vivo production of the nematode Thripinema nicklewoodi (Siddiqi), an obligate parasite and potential biological control agent of western flower thrips Frankliniella occidentalis (Pergande). Nematode infection is not lethal but causes sterilization of adult female hosts. Both fertilization and horizontal transmission of T. nicklewoodi is achieved in 1.5-ml microcentrifuge tubes (infection arenas), in the presence of 100% humidity, a temporary food source and preferably a damp substrate. Following exposure to infection arenas, F. occidentalis are reared on excised bean leaves Phaseolus vulgaris (L.) in polypropylene containers for 2 wk at 25°C to allow the reproduction and development of a single generation of nematodes within infected hosts’s abdominal cavity. To identify infected hosts after this incubation period, thrips are isolated in microcentrifuge tubes and monitored for free-living nematodes being released along with frass. Infected thrips are reintroduced back into infection arenas to inoculate further thrips to maintain the culture. We documented the output of the rearing procedure using a standard method and following simple manipulation of several individual parameters of the infection technique. The standard method was the most efficient, and resulted in an increased (output/input) ratio of infected thrips of ≈2; i.e., the number of infected thrips approximately doubles each generation. Monitoring infected thrips revealed that nematodes were first released between 12–14 d postinfection and for an average of 7.9 d at 25°C; highlighting the potential to reuse infective thrips between infection arenas. The possibility of using T. nicklewoodi as an inoculative agent against F. occidentalis infesting floricultural crops is discussed.