G-protein-coupled receptors (GPCRs), one of the most versatile groups of cell surface receptors, can recognize specific ligands from neural, hormonal, and paracrine organs and regulate cell growth, proliferation, and differentiation. Gpr48/LGR4 is a recently identified orphan GPCR with unknown functions. To reveal the functions of Gpr48 in vivo, we generated Gpr48 ؊/؊ mice and found that Gpr48 ؊/؊ fetuses displayed transient anemia during midgestation and abnormal definitive erythropoiesis. The dramatic decrease of definitive erythroid precursors (Ter119 pos population) in Gpr48 ؊/؊ fetal liver at E13.5 was confirmed by histological analysis and blood smear assays. Real-time PCR analyses showed that in Gpr48؊/؊ mice both adult hemoglobin ␣ and ␤ chains were decreased while embryonic hemoglobin chains (, ␤H1, and ⑀y) were increased, providing another evidence for the impairment of definitive erythropoiesis. Furthermore, proliferation was suppressed in Gpr48 ؊/؊ fetal liver with decreased c-Myc and cyclin D1 expression, whereas apoptosis was unaffected. ATF4, a key transcription factor in erythropoiesis, was downregulated in Gpr48 ؊/؊ fetal livers during midgestation stage through the cAMP-PKA-CREB pathway, suggesting that Gpr48 regulated definitive erythropoiesis through ATF4-mediated definitive erythropoiesis.Erythropoiesis occurs sequentially in distinct anatomical locations in two different phases during embryogenesis. The earlier phase is defined as primitive erythropoiesis, which, in the mouse, originates from the yolk sac at embryonic day 7.5 (E7.5), 3 and the later phase is definitive erythropoiesis, which was carried out in the fetal liver during midgestation after E12.5. Non-nucleated adult-type red blood cells are first generated in the fetal liver, the primary organ for erythropoiesis during midgestation from E12 to E16 (1). Erythropoiesis then transfers to bone marrow and spleen in the adult (2, 3). However, the molecular mechanism of regulating erythropoiesis has not been completely delineated. Most of the studies were focused on the transcription factors to explore the mechanisms of erythropoiesis (4, 5). In recent years, several transcription factors were also identified to regulate definitive erythropoiesis in fetal liver during midgestation (6 -11). One of the transcription factors is ATF4, which has been shown to regulate cell proliferation in response to a broad spectrum of cell stresses and can be either an activator or a repressor in response to different extracellular signals (12). ATF4 Ϫ/Ϫ mice have been reported to cause defective definitive erythropoiesis, and severe anemia at midgestation (13). Although receptors, such as c-Kit and EPOR, have been well studied in erythropoiesis (14), little is known on the function of G-proteincoupled receptors (GPCRs) in erythropoiesis during development (14, 15).The GPCR family represents the largest and most versatile group of cell surface receptors (16 -18). GPCRs can recognize their ligands, a diverse array of extracellular signals, then transmit th...