Fluorescent proteins (FPs) are commonly used to label specific cell populations and molecules in model organisms, but quantifying the intrinsic and extrinsic variables that influence experimental outcomes has been difficult. Chen et al. generate eight different FP-expressing alleles and express them in mouse embryonic stem cells or in mice to assess the contribution of different variables on FP expression levels and brightness. Their findings reveal that outcomes are markedly influenced by genetic factors present in the allele as well as the type of FP. Furthermore, in vivo analysis of Cherry-expressing mice shows up to 30-fold variation in expression between different tissue types.