Although tick-borne infectious diseases threaten human and animal health worldwide, with constantly increasing incidence, little knowledge is available regarding vector–pathogen interactions and pathogen transmission. In vivo laboratory study of these subjects using live, intact ticks is expensive, labor-intensive, and challenging from the points of view of biosafety and ethics. Several in vitro models have been developed, including over 70 continuous cell lines derived from multiple tick species and a variety of tick organ culture systems, facilitating many research activities. However, some limitations have to be considered in the translation of the results from the in vitro environment to the in vivo situation of live, intact ticks, and vertebrate hosts. In this review, we describe the available in vitro models and selected results from their application to the study of tick-borne viruses, bacteria, and protozoa, where possible comparing these results to studies in live, intact ticks. Finally, we highlight the strengths and weaknesses of in vitro tick culture models and their essential role in tick-borne pathogen research.