Over the past decades, major efforts were undertaken to develop devices on a nanoscale level for the efficient and nontoxic delivery of molecules to tissues and cells, for the purpose of either diagnosis or treatment of disease. The application of such devices in drug delivery has proven to be beneficial for matters as diverse as drug solubility, drug targeting, controlled drug release, and transport of drugs across cellular barriers. Multiple nanotherapeutics have been approved for clinical treatment, and more products are being evaluated in preclinical and clinical trials. However, many biological barriers hinder the medical application of nanocarriers. There are two main classes of barriers that need to be overcome by drug nanocarriers: extracellular and intracellular barriers, both of which may capture and/or destroy therapeutics before they reach their target site. This Account discusses major biological barriers that are confronted by nanotherapeutics, following their systemic administration, focusing on cellular entry and endosomal escape of gene delivery vectors. The use of pH-responsive materials to overcome the endosomal barrier is addressed. Historically, cell biologists have studied the interaction between cells and pathogens in order to unveil the mechanisms of endocytosis and cell signaling. Meanwhile, it is becoming clear that cells may respond in similar ways to artificial drug delivery systems and, consequently, that knowledge on the cellular response against both pathogens and nanoparticulate systems will aid in the design of improved nanomedicine. A close collaboration between bioengineers and cell biologists will promote this development. At the same time, we have come to realize that tools that we use to study fundamental cellular processes, including metabolic inhibitors of endocytosis and overexpression/downregulation of proteins, may cause changes in cellular physiology. This calls for the implementation of refined methods to study nanocarrier-cell interactions, as is discussed in this Account. Finally, recent papers on the dynamics of cargo release from endosomes by means of live cell imaging have significantly advanced our understanding of the transfection process. They have initiated discussion (among others) on the limited number of endosomal escape events in transfection, and on the endosomal stage at which genetic cargo is most efficiently released. Advancements in imaging techniques, including super-resolution microscopy, in concert with techniques to label endogenous proteins and/or label proteins with synthetic fluorophores, will contribute to a more detailed understanding of nanocarrier-cell dynamics, which is imperative for the development of safe and efficient nanomedicine.