Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects. To understand the cause of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells. Here we describe a simple protocol using highly soluble and fixable dye called LysoTracker to look at PCD in mouse embryos null for the growth factor, sonic hedgehog. In addition, we demonstrate how to analyze PCD using LysoTracker in differentiating ES cell cultures. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD in tissues and differentiating ES cells.