Understanding of reproductive biology in canids, including the domestic dog, is surprisingly limited. This includes the regulators of ovarian follicle development, and mechanisms of anestrus termination, fertilization and embryo development. In turn, this lack of understanding has limited our ability to develop assisted reproductive technologies (ART) for endangered canid conservation efforts. ART of interest include in vitro follicle culture for maternal genome rescue, estrus induction protocols, and in vitro fertilization (IVF). Here, we describe: 1) Studies evaluating the stage-specific requirements for follicle stimulating hormone (FSH), luteinizing hormone (LH), and activin on domestic dog follicle development in vitro. We demonstrate the beneficial effects of FSH and activin on growth, and activin on antrum expansion and oocyte health in short term culture. 2) Evaluation of serum collected during the anestrus to estrus transition revealing a significant increase in anti-Müllerian hormone (AMH) during proestrus, likely originating from increased numbers of antral follicles during this time. 3) The birth of the first live puppies from IVF embryos utilizing in vivo matured oocytes. Further, consistently high rates of embryo production are obtained using the described system, with no effect of progesterone supplementation to embryo culture media. Improved knowledge of the drivers of follicle development inform understanding of the resumption of estrus cyclicity, which in turn is needed to explore mechanisms of oocyte maturation prior to fertilization. In vitro fertilization is also required to assess the developmental capacity of oocytes grown from cultured follicles. Thus, these combined discoveries represent significant advancements in the understanding of canid reproductive biology and potential application to development of ART for the management of biomedical research colonies and for endangered canid conservation.