In vivo, high-resolution, time-lapse imaging characterized lens development in the zebrafish from 16 to 96 hr postfertilization (hpf). In zebrafish, the lens placode appeared in the head ectoderm, similar to mammals. Delamination of the surface ectoderm resulted in the formation of the lens mass, which progressed to a solid sphere of cells separating from the developing cornea at approximately 24 hpf. A lens vesicle was not observed and apoptosis was not a major factor in separation of the lens from the future cornea. Differentiation of primary fibers began in the lens mass followed by formation of the anterior epithelium after delamination was complete. Secondary fibers differentiated from elongating epithelial cells near the posterior pole. Quantification characterized three stages of lens growth. The study confirmed the advantages of live-cell imaging for three-dimensional quantitative structural characterization of the mechanism(s) responsible for cell differentiation in formation of a transparent, symmetric, and refractile lens. Developmental Dynamics 238:2254–2265, 2009. © 2009 Wiley-Liss, Inc.