In females, reproduction is a key facet of women’s health, livestock production, and conservation of endangered species. The ovary is the gonad of the female and is a central component of female reproductive physiology as it houses the germ cells (i.e., oocytes) within follicles and produces hormones to coordinate folliculogenesis. The ovary is a target organ for developing novel assisted reproductive technologies (ART) such as safeguarding fertility via cryopreserving ovarian tissue containing follicles and developing strategies to grow early staged preantral follicles in vitro for oocyte production. Moreover, a better understanding of cellular and molecular events constituting early gonad development would be widely valuable to further advance ART that capitalizes on components of the ovary. In this work, we describe three aims using the bovine as a model and collectively expand our current knowledge in 1) ovarian tissue cryopreservation (OTC), 2) in vitro bovine preantral follicle development, and 3) in vitro differentiation of bovine embryonic stem cells (bESCs) into progenitors of bipotential gonad-like cells. In Aim 1, we found that, overall, slow freezing outperformed vitrification when bovine ovarian fragments were thawed and cultured as indicated by higher rates of normal follicle morphology, lower rates of apoptosis in stromal cells, and proper expression of the gap junction protein connexin 37. In addition to the use of OTC and culturing ovarian tissue fragments to rescue folliculogenesis, in Aim 2 we explored the use of bioengineered and proteolytically degradable (poly)ethylene glycol (PEG) hydrogels for in vitro culture of isolated bovine preantral follicles. We also tested co-encapsulation of mesoderm-like cells (MeLCs) from bESCs or native bovine ovarian cells (BOCs) with follicles to determine their propensity to become theca-like cells and improve folliculogenesis in vitro. After customizing a PEG hydrogel to complement bovine preantral follicle extracellular matrix (ECM) enzyme expression, we found that smaller follicles grew better in PEG hydrogels compared to two-dimensional control over ten days. However, MeLCs did not survive PEG hydrogel encapsulation process. Although BOCs could survive in PEG hydrogels throughout the culture period, BOCs did not better support follicle survival and development when co-encapsulated compared to PEG-only control. Moreover, the culture conditions did not maintain expression of selected genes essential for theca cell differentiation. Finally, in Aim 3, we tested the ability of bESCs to be driven towards the intermediate mesoderm (IM), early coelomic epithelium (eCE), and steroidogenic state during in vitro culture using several protocols. Our data indicated that inclusion of the steroidogenic factor-1 (SF1) agonist, RJW100, did not upregulate steroidogenic genes during the differentiation. Similarly, the cytokines basic fibroblast growth factor (bFGF) and bone morphogenic protein-4 (BMP4) were dispensable for inducing IM and eCE gene expression, and WNT activation was a predominant driver of eCE and IM gene expression, high concentrations of BMP4 led to upregulation of lateral plate mesoderm gene expression, and paraxial mesoderm gene expression remained unchanged across all regimens. Collectively, this dissertation work sheds light on several facets of ovarian biology including a better understanding of the impacts of OTC, in vitro preantral folliculogenesis, and use of embryonic stem cells to recapitulate bipotential gonad somatic cell differentiation. Nevertheless, additional research is required to elucidate mechanisms that are pivotal to both early folliculogenesis and early embryonic gonad formation in the bovine model.