The unique spectral properties and versatility of autofluorescent proteins have facilitated their widespread use in flow cytometric applications. The ability to analyze heterologous fluorescent protein expression conveniently and noninvasively by individually interrogating cells has facilitated increasingly more sophisticated experimental designs to address important biological questions. Improved multilaser flow cytometers have allowed the fluorescent protein field to flourish by permitting high-speed, multiparametric analysis of biological samples. Fluorescent proteins are well suited for either transient or stable expression analysis. Therefore, achieving efficient gene transfer and expression in cells by transfection or viral transduction is paramount to the optimal use of fluorescent proteins in flow cytometry. The archetypal autofluorescent protein, enhanced green fluorescent protein (eGFP), can be used successfully in combination with other fluorescent protein variants. Two such variants, Cerianthus sp. orange fluorescent protein (cOFP) and a fast maturing variant of Discosoma sp. red protein (DsREDExpress), are well suited for flow cytometric applications in combination with eGFP and do not require special filters for optimal excitation and detection.