Serotonergic neurons project widely throughout the CNS and modulate many different brain functions. Particularly important, but controversial, are the contributions of serotonin (5-HT) neurons to respiratory and thermoregulatory control. To better define the roles of 5-HT neurons in breathing and thermoregulation, we took advantage of a unique conditional knock-out mouse in which Lmx1b is genetically deleted in Pet1-expressing cells (Lmx1b^f/f/p), resulting in near-complete absence of central 5-HT neurons. Here, we show that the hypercapnic ventilatory response in adult Lmx1b^f/f/p mice was decreased by 50% compared with wild-type mice, whereas baseline ventilation and the hypoxic ventilatory response were normal. In addition, Lmx1b^f/f/p mice rapidly became hypothermic when exposed to an ambient temperature of 4°C, decreasing core temperature to 30°C within 120 min. This failure of thermoregulation was caused by impaired shivering and nonshivering thermogenesis, whereas thermosensory perception and heat conservation were normal. Finally, intracerebroventricular infusion of 5-HT stimulated baseline ventilation, and rescued the blunted hypercapnic ventilatory response. These data identify a previously unrecognized role of 5-HT neurons in the CO₂ chemoreflex, whereby they enhance the response of the rest of the respiratory network to CO₂. We conclude that the proper function of the 5-HT system is particularly important under conditions of environmental stress and contributes significantly to the hypercapnic ventilatory response and thermoregulatory cold defense.