This chapter describes the progress that has been made in the development of in vitro ovarian follicle culture systems, and highlights the importance of maintaining follicular architecture to facilitate follicle growth and oocyte development. The follicle is the main reproductive unit of the ovary, and is composed of a single oocyte and its surrounding somatic support cells. The interaction between follicle cells and the surrounding extracellular matrix (ECM) is essential for facilitating intercellular communication within an individual follicle. The goal in ovarian follicle culture, particularly with follicles from larger mammalian species, is to mimic the complex molecular, functional, and structural changes that occur in vivo in order to produce a fully mature, fertilizable egg in vitro. The chapter reviews the various systems that have been developed for the in vitro culture of preantral follicles. Initial work focused on achieving oocyte growth in vitro relied on adherent two-dimensional (2D) culture systems.