Innate vocalizations play a critical communicative role for both human and non-human animals. Infants of altricial species vocalize when hungry, cold, or isolated from caregivers and their nest environment. These ‘infant vocalizations’ are crucial for the survival of altricial infants, including humans, as they attract caregiver attention. As altricial infants gradually develop the skills to survive independently, they stop producing infant vocalizations and begin producing adult-like vocalizations in a variety of behavioral contexts, including during courtship and affiliative interactions. Although this developmental transition in vocal communication is ubiquitous across mammals, the brain mechanisms that enable the transition from infant vocalizations to adult vocalizations remain largely unknown. Studying the ontogeny of vocal communication in laboratory mice (Mus musculus) provides a tractable opportunity to begin to understand the behavioral and neural mechanisms that underlie this transition in vocal communication. A wide range of transgenic tools are available in mice, offering a unique opportunity to measure and manipulate activity in vocalization-related brain regions. Furthermore, mouse ultrasonic vocalizations (i.e., USVs) exhibit a pronounced developmental transition. Infant mice produce USVs in response to cold and isolation from the nest (i.e., isolation USVs). As pups become less dependent on caregivers for survival during early development, their rates of isolation USVs decline and reach near-zero rates by approximately the third week of postnatal life. Adult female and male mice, in contrast, produce USVs when interacting with same-sex and opposite-sex conspecifics (i.e., social USVs) and produce very few USVs when recorded alone. How the brain regulates this developmental transition in vocal communication remains largely unknown. In this body of work, I present three studies addressing the various gaps in our knowledge of mouse vocal communication outlined above. In Chapter 2, I describe vocal ontogeny in infant mice by longitudinally tracking isolation USVs and non-vocal behaviors at postnatal days 5, 10, 15, and 20. In this work, I explore the extent to which non-vocal behaviors of mouse pups relate to the within-age variability in rates and acoustic features of isolation USVs. I use machine-learning based behavior quantification tools and uncover a previously unknown relationship between mouse isolation USV rates and non-vocal behaviors during early development. These results demonstrate the importance of moving away from viewing infant vocalizations as a standalone behavior and to instead consider them in the context of intrinsic and extrinsic factors that might regulate their production. In Chapter 3, I describe the developmental timing of the emergence of mouse social USVs. I show that juvenile mice begin producing their initial social USVs earlier than previously reported, between postnatal days 20-24. These early social USVs are also temporally coordinated with active social interaction, as is the case in adults. Findings from Chapter 3 contribute to the existing literature on mouse vocal development during adolescence and set the stage for future studies to understand brain mechanisms that underlie developmental changes in vocal communication. In Chapter 4, I use the behavioral findings from Chapters 2 and 3 as a foundation to begin exploring such brain mechanisms. I focus on a part of the brain that has been implicated in innate vocalizations of vertebrates, including adult mouse social USVs - the midbrain periaqueductal gray (PAG). In Study 4a, I begin testing the hypothesis that the same PAG neurons regulate USV production across development, in both pups and adults. I use immunohistochemistry to detect expression of Fos, a marker for recent neural activity, in the PAG of pups following the production of isolation USVs and in adolescent mice following the production of social USVs. I report elevated Fos expression in the PAG of both pups and adolescents following USV production. The numbers of Fos-positive PAG neurons in both age groups were also positively correlated to USV rates. These results reveal correlational evidence suggesting that the PAG regulates pup isolation USVs and adolescent social USVs. In Study 4b, I develop and apply an experimental protocol in adult mice that incorporates a short viral expression wait time and that successfully implements chemogenetic activation of glutamatergic PAG neurons to elicit USV production in adult females and males. This protocol, in turn, can be used in future work to test the involvement of these PAG neurons in regulating the production of pup isolation USVs.