The histone demethylase Jmjd3 plays a critical role in cell lineage specification and differentiation at various stages of development. However, its function during normal myeloid development remains poorly understood. Here, we carried out a systematic in vivo screen of epigenetic factors for their function in hematopoiesis and identified Jmjd3 as a new epigenetic factor that regulates myelopoiesis in zebrafish. We demonstrated that jmjd3 was essential for zebrafish primitive and definitive myelopoiesis, knockdown of jmjd3 suppressed the myeloid commitment and enhanced the erythroid commitment. Only overexpression of spi1 but not the other myeloid regulators rescued the myeloid development in jmjd3 morphants. Furthermore, preliminary mechanistic studies demonstrated that Jmjd3 could directly bind to the spi1 regulatory region to alleviate the repressive H3K27me3 modification and activate spi1 expression. Thus, our studies highlight that Jmjd3 is indispensable for early zebrafish myeloid development by promoting spi1 expression. Copyright © 2018 Elsevier B.V. All rights reserved.