Eukaryotic cells use actin polymerization to change shape, move, and internalize extracellular materials by phagocytosis and endocytosis, and to form contractile structures. In addition, several pathogens have evolved to use host cell actin assembly for attachment, internalization, and cell-to-cell spread. Although cells possess multiple mechanisms for initiating actin polymerization, attention in the past five years has focused on the regulation of actin nucleation-the formation of new actin filaments from actin monomers. The Arp2/3 complex and the multiple nucleation-promoting factors (NPFs) that regulate its activity comprise the only known cellular actin-nucleating factors and may represent a universal machine, conserved across eukaryotic phyla, that nucleates new actin filaments for various cellular structures with numerous functions. This review focuses on our current understanding of the mechanism of actin nucleation by the Arp2/3 complex and NPFs and how these factors work with other cytoskeletal proteins to generate structurally and functionally diverse actin arrays in cells.