Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1−/−) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT2A and 5-HT2B receptors toward terminal differentiation. In addition, red blood cells from 5-HT–deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1−/− animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT–deficient and normal cells. Our thorough analysis of TPH1−/− mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.