Stage-specific embryonic antigens (SSEAs) are cell-surface molecules that exhibit lineage-restricted patterns of expression during development. These antigens provide useful markers for identifying embryonic stem cells and their differentiated derivatives. SSEA-1 provides a surface marker for mouse and human primordial germ cells and mouse embryonic stem cells. SSEA-3 and SSEA-4 provide surface markers for human embryonic stem cells. Here, we describe methods for staining embryonic stem cells in culture and suspension using specific primary antibodies and fluorescent dye- or enzyme-conjugated secondary antibodies. Methods for alkaline phosphatase staining and immunohistochemical localization of antigens in paraffin sections are also described. Monoclonal antibodies directed to SSEAs are valuable research tools that can be used to: (1) determine the percentage of stem cells in mixed cultures; (2) examine the spatial distribution of stem cells and differentiated derivatives in monolayer cultures and embryoid bodies; and (3) isolate populations of differentiated and undifferentiated cells by immunomagnetic or fluorescence-activated cell sorting for cellular and molecular analyses.