Introduction: Feedback control systems that regulate breathing require a flexible, dynamic system capable of responding to acute ventilatory challenges including hypercapnia and acidosis. This reflex arc must also be adaptable to additional environmental changes, including shifts in ambient temperature. Brainstem serotonergic (5-HT) neurons play integral roles in acute arterial CO2/pH regulation, but also participate in cold defense mechanisms and facilitate thermogenesis. How is the CO2/pH chemoreflex altered in mammals with varying environmental or brain temperature? And, are there cellular correlates to these unique interactions of temperature and CO2 chemoreception? Methods: We study breathing using whole body plethysmography, and thermoregulation with a variety of thermal imaging and temperature detection techniques. These were applied to genetically modified mouse models of 5-HT neuron lesions and/or dysfunction. Results: Moderate or near-complete genetic deletion of central 5-HT-producing neurons in conscious mice leads to selective but severe deficits in ventilatory responses to hypercarbia, and failure to generate sufficient heat to defend core body temperatures during a cold challenge. Mild environmental cooling in these models led to small but significant drops in core temperature which negatively affected both the hypercapnic and hypoxic ventilatory responses, nearly exclusively in male but not female mice. Infusing additional 5-HT into the CNS “rescued” the CO2 chemoreflex but failed to alter cold defense failure. Direct cellular recordings identified a binary phenotype – 5-HT neurons either respond to increased temperature or CO2/pH directly in acute slices from young mice. Finally, knockout of CNS 5-HT in rats, while retaining the neurons that would otherwise make 5-HT, has no effects on either the CO2 chemoreflex or thermoregulation in conscious rats. Conclusions: We conclude that brainstem neurons that synthesize both at the cellular and organismal level contribute to defenses against hypercapnia and acidosis and cold environments, but this may relate to additional neurotransmitters produced by these neurons and not 5-HT itself.