Adult zebrafish stripes are formed from stripes of gold iridophores alternating with stripes of black melanocytes lying beneath silver stripes. Analysis of defects in pigment pattern development caused by sparse (spa, rose (ros), and leopard (leo) single and double mutant combinations suggests that spa+ and ros+ functions are required for development of separate populations of pigment cells in the adult and that leo+ functions to control assembly of melanocytes into stripes. Thus, between 2 and 3 weeks of zebrafish development, spa-dependent melanocytes differentiate throughout the flank, followed by leo-dependent assembly of these cells into stripes. Beginning at 3 weeks of development, a distinct ros-dependent population of melanocytes differentiates in the stripe. Both early and late differentiating melanocytes then affect the formation of the silver stripes, ensuring registration of melanocyte and iridophore stripes.