Actin filament assembly and turnover drive many forms of cellular motility, particularly extension of the leading edge of locomoting cells and rocketing of pathogenic microorganisms through host cell cytoplasm. De novo nucleation of actin filaments appears to be required for these movements. A complex of seven proteins called Arp2/3 complex is the best characterized cellular initiator of actin filament nucleation. Arp2/3 complex is intrinsically inactive, relying on nucleation promoting factors for activation. WASp/Scar family proteins are prominent cellular nucleation promoting factors. They bring together an actin monomer and Arp2/3 complex in solution or on the side of an existing actin filament to initiate a new filament that grows in the barbed end direction. WASp and N-WASP are intrinsically autoinhibited, and their activity is regulated by Rho-family GTPases such as Cdc42, membrane polyphosphoinositides, WIP/verprolin, and SH3 domain proteins. These interactions provide a final common pathway for many signaling inputs to regulate actin polymerization. Microorganisms either activate Arp2/3 complex directly or usurp N-WASP to initiate actin polymerization.