The ETS protein Spi-1/Pu.1 plays a pivotal and widespread role throughout hematopoiesis in many species. This study describes the identification, characterization and functional analysis of a new zebrafish spi transcription factor spi-1-like (spi-1l) that is expressed in primitive myeloid cells, erythro-myelo progenitor cells, and in the adult kidney. Spi-1l functions genetically downstream of etsrp, scl, and spi-1/pu.1 in myeloid differentiation. Spi-1l is co-expressed in a subset of spi-1/pu.1 cells and its function is necessary and sufficient for macrophage and granulocyte differentiation. These results establish a critical role for spi-1l in zebrafish myeloid cell differentiation.