Embryonic stem cells (ESCs) have the most pluripotent potential of any stem cell. These cells, isolated from the inner cell mass of the blastocyst, are "pluripotent," meaning that they can give rise to all cell types within the developing embryo. As a result, ESCs have been regarded as a leading candidate source for novel regenerative medicine therapies and have been used to derive diverse cell populations, including myocardial and endothelial cells. However, before they can be safely applied clinically, it is important to understand the in vivo behavior of ESCs and their derivatives. In vivo analysis of ESC-derived cells remains critically important to define how these cells may function in novel regenerative medicine therapies. In this review, we describe several available imaging modalities for assessing cell engraftment and discuss their strengths and limitations. We also analyze the applications of these modalities in assessing the utility of ESCs in regenerative medicine therapies.