The generation of tissue-specific mouse models has provided a powerful strategy to understand the role of genes in specific tissues/cells of interest under control/basal conditions and in response to physiological and pathological stimuli. Here we describe the generation of cardiomyocyte-specific FoxO1 knockout mice using Cre-loxP technology to examine the role of FoxO1 for the induction of heart enlargement (cardiac hypertrophy) in settings of health and disease. We highlight breeding strategies for generating tissue-specific mouse models and key experimental considerations during characterization.