PGE(2) is involved in a wide variety of physiological and pathological processes; however, deciphering its role in early mammalian development has been difficult due to the maternal contribution of PGE(2). To overcome this limitation we have investigated the role of PGE(2) during T cell development in zebrafish. In this study, we show that zebrafish ep4a, a PGE(2) receptor isoform of EP4, is expressed at 26 h postfertilization in the dorsal aorta-posterior cardinal vein joint region, which has a high homology with the mammal aorta-gonad-mesonephros area and where definitive hemopoiesis arises. Furthermore, it is expressed in the presumptive thymus rudiment by 48 h postfertilization. Supplementation of PGE(2) results in a strong increase in rag1 levels and cell proliferation in the thymus. In contrast, the inhibition of PGE(2) production, as well as EP4 blockade, abrogates the expression of rag1 in the thymus and that of the lymphoid precursor marker ikaros, not only in the dorsal aorta-posterior cardinal vein joint region but also in the newly identified caudal hemopoietic tissue without affecting early hemopoietic (scl, gata2) and erythropoietic (gata1) markers. These results identify ep4a as the earliest thymus marker and define a novel role for the PGE(2)/EP4 pathway in controlling T cell precursor development in zebrafish.