The use of mouse derived mammary organoids can provide a unique strategy to study mammary gland development across a normal life cycle, as well as offering insights into how malignancies form and progress. Substantial cellular and epigenomic changes are triggered in response to pregnancy hormones, a reaction that engages molecular and cellular changes that transform the mammary epithelial cells into "milk producing machines". Such epigenomic alterations remain stable in post-involution mammary epithelial cells and control the reactivation of gene transcription in response to re-exposure to pregnancy hormones. Thus, a system that tightly controls exposure to pregnancy hormones, epigenomic alterations, and activation of transcription will allow for a better understanding of such molecular switches. Here, we describe the characterization of ex vivo cultures to mimic the response of mammary organoid cultures to pregnancy hormones and to understand gene regulation and epigenomic reprogramming on consecutive hormone exposure. Our findings suggest that this system yields similar epigenetic modifications to those reported in vivo, thus representing a suitable model to closely track epigenomic rearrangement and define unknown players of pregnancy-induced development.