Selective marking of a single cell within an embryo is often difficult to perform with existing methods. Here, we report a minimally invasive optical technique that uses 405-nm laser light to photoactivate a variant of green fluorescent protein (PAGFP). Single cells and small groups of cells (n < 10) are successfully marked, from a region of cells injected and electroporated with PAGFP, in both whole chick embryo explants and in ovo. Photoactivated cells display normal cell migratory behaviors and retain a bright GFP signal for at least 24 hr when followed with confocal time-lapse microscopy. We determined that using a low-magnification objective (∼ ×10) and low laser power (∼1–10%) leads to a steady increase in fluorescence signal within a photoactivated cell and minimizes photobleaching. The utility of PAGFP photoactivation was tested to address a specific question in developmental biology. Specifically, we asked whether neighboring migratory cells that emerge from the hindbrain and invade surrounding peripheral tissues maintain neighbor relationships while traveling to the destination sites. We found that some neural crest do not maintain neighbor relationships, such that two neighboring cells near the neural tube cells may populate different branchial arches. The ability to optically photoactivate PAGFP in a single or small group of cells and follow individual cell migratory behaviors within a living embryo offers a powerful, minimally invasive cell marking tool for precise, in vivo cell migration studies. Developmental Dynamics 233:983–992, 2005. © 2005 Wiley-Liss, Inc.