This chapter reviews in ovo vaccination of chicken eggs. It first discusses the history and evolution of in ovo vaccination, then goes on to highlight the various interacting factors of in ovo vaccination success. A section on additional interactions and considerations are also provided, focusing specifically on egg candling for viability, chick handling and welfare, labor efficiencies and quality assurance. The chapter concludes by providing an overview of the challenges that need to be overcome in order to develop more efficient vaccines for in ovo use.