Publisher Summary This chapter focuses on the use of stable transgenic zebrafish lines, expressing green fluorescent protein (GFP) under the control of the pu.1 promoter, to examine myeloid cell differentiation and development. The optically clear zebrafish embryo is particularly amenable to in vivo analyses and the use of GFP expression for monitoring hematopoietic development in transgenic zebrafish. The relative stability of the GFP protein allows it to be used as a cell tracking marker to experimentally analyze hematopoietic cell differentiation and movements, correlated with developmental changes in gene expression patterns. Zebrafish and mammals share many of the morphological and cytological features of myelopoietic cell types, including granulocytes and monocyte/macrophages. The PU.1 protein belongs to the ets family of transcription factors and plays an early role in myelopoiesis that is essential for the development of both myeloid (granulocytes and monocytes/macrophages) and lymphoid cells. Development of a transgenic zebrafish expressing EGFP under control of the zebrafish pu.1 promoter is a useful tool to study the regulation of myeloid cell differentiation in vivo. Stable transgenic zebrafish lines specifically expressing EGFP in subsets of hematopoietic cells can play an important role in elucidating the mechanisms that regulate vertebrate hematopoiesis. Because embryonic zebrafish is amenable to experimental manipulations, such as transplantation and targeted gene knockdown assays, questions of hematopoietic stem and progenitor cell regulation and lineage determination can be addressed in vivo. The ability to sort these living cells by fluorescence allows their morphological and genetic analysis and transplantation studies by using the purified population of cells isolated at different stages of development.