The mammalian transcription factor SPI-1 (synonyms: SPI1, PU.1, or Sfpi1) plays a critical role in myeloid development. To examine early myeloid commitment in the zebrafish embryo, we isolated a gene from zebrafish that is a SPI-1 orthologue on the basis of homology and phylogenetic considerations. The zebrafish spi1 (pu1) gene was first expressed at 12 h postfertilization in rostral lateral plate mesoderm (LPM), anatomically isolated from erythroid development in caudal lateral plate mesoderm. Fate-mapping traced rostral LPM cells from the region of initial spi1 expression to a myeloid fate. spi1 expression was lost in the bloodless mutant cloche, but rostral spi1 expression and myeloid development were preserved in the mutant spadetail, despite its complete erythropoietic failure. This dissociation of myeloid and erythroid development was further explored in studies of embryos overexpressing BMP-4, or chordin, in bmp-deficient swirl and snailhouse mutants, and chordin-deficient chordino mutants. These studies demonstrate that, in zebrafish, spi1 marks a rostral population of LPM cells committed to a myeloid fate anatomically separated from and developmentally independent of erythroid commitment in the caudal LPM. Such complete anatomical and developmental dissociation of two hematopoietic lineages adds an interesting complexity to the understanding of vertebrate hematopoietic development and presents significant implications for the mechanisms regulating axial patterning. © 2002 Elsevier Science (USA).