To investigate flexibility in vocal signaling by rodent pups, we examined whether olfactory stimuli influence characteristics of pup calls and how these calls may be affected by sex and litter size in California mice (Peromyscus californicus). Pups were isolated and recorded during a 3-minute baseline period followed by a 5-minute exposure to bedding containing scent from their home cage, scent from the home cage of an unfamiliar family, coyote urine, or no scent (control). Latency to call, call rate, and call characteristics (duration, frequency, and amplitude) were compared between the baseline and scent-exposure periods and among olfactory conditions. Compared to the control condition, pups from 2-pup litters called more quietly when exposed to odor from a predator, while pups from 3-pup litters called more loudly. Additionally, pups showed non-significant tendencies to reduce call rates in response to odors from their home cage and to increase call rates when exposed to predator urine. Lastly, males produced higher-frequency calls and more ultrasonic vocalizations than females. These results indicate that pup calling behavior in this species can be influenced by acute olfactory stimuli as well as litter size and sex. The flexibility of pup calling in response to these three variables potentially increases the communication value of pup calls and help shape the parents’ responses.