The differentiation of hematopoietic stem cells (HSCs) into distinct blood lineages is orchestrated by a tight regulation of critical genes during development. To describe the genetic program during the early steps of blood and blood vessel formation in greater detail, we analyzed the expression profiles of zebrafish cloche mutants (clo), in which blood and endothelial cells are completely abrogated, and of isolated hematopoietic and vascular progenitor cell populations from FAC-sorted transgenic lmo2-GFP zebrafish embryos. Consistent with a defect in hemangioblast differentiation, clo mutants at the 5 somite stage were characterized by a prominent downregulation of transcription factors, such as scl and lmo2. As a novel finding, clo mutants at this stage strongly upregulated muscle-related genes, such as myosin, actin, or creatine kinase. FAC-sorted lmo2-GFP cells from 14 somite stage embryos expressed similar hematopoietic transcription factors including scl, lmo2, and gata1. In addition, socs genes, tgfb, fos and jun related genes, and heat shock proteins were also strongly expressed. Comparison with the expression profile of human and mouse fetal liver HSCs revealed a similar regulation between fish and mammals: Expression of 102/168 genes from lmo2-GFP positive cells were also present in mammalian fetal HSCs, indicating an evolutionary highly conserved genetic program of blood formation. In sum, gene expression profiling of zebrafish mutants and of sorted progenitor cells from transgenic zebrafish represent a powerful and versatile tool to define the genetic regulation of vertebrate hematopoietic development.