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The molecular and cellular mechanisms that underlie the many roles of macrophages in health and disease states in vivo remain poorly understood. The purpose of this Review is to present and discuss current knowledge on the developmental biology of macrophages, as it underlies the concept of a layered myeloid system composed of 'resident' macrophages that originate mainly from progenitor cells generated in the yolk sac and of 'passenger' or 'transitory' myeloid cells that originate and renew from bone marrow hematopoietic stem cells, and to provide a framework for investigating the functions of macrophages in vivo.