The Spi1/Pu.1 transcription factor plays a crucial role in myeloid cell development in vertebrates. Despite extensive studies of Spi1 the controlled gene group remains largely unknown. To identify genes dependent on Spi1 we employed a microarray strategy using a knockdown approach in zebrafish embryos combined with FACS sorting of myeloid cells from transgenic embryos. This approach of using knockdowns with specific GFP-marked cell types showed highly successful to identify macrophage-specific genes in Spi1-directed innate immunity. We found a gene group down-regulated upon spi1 knockdown, which is also enriched in FACS-sorted embryonic myeloid cells of a spi1:GFP transgenic line. This gene group, representing putative myeloid-specific Spi1 target genes, contained all five previously identified Spi1-dependent zebrafish genes, as well as a large set of novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes cxcr3.2, mpeg1, ptpn6 and mfap4 were expressed specifically in early embryonic macrophages. In a functional approach we demonstrated that gene cxcr3.2, coding for chemokine receptor 3.2, is involved in macrophage migration to the site of bacterial infection. Therefore, based on our combined transcriptome analyses, we discovered novel early macrophage-specific marker genes, including a signal transducer pivotal for macrophage migration in the innate immune response.