Understanding transmission biology at an individual level is a key component of intervention strategies that target the spread of malaria parasites from human to mosquito. Gametocytes are specialized sexual stages of the malaria parasite life cycle developed during evolution to achieve crucial steps in transmission. As sexual differentiation and transmission are tightly linked, a deeper understanding of molecular and cellular events defining this relationship is essential to combat malaria. Recent advances in the field are gradually revealing mechanisms underlying sexual commitment, gametocyte sequestration, and dynamics of transmissible stages; however, key questions on fundamental gametocyte biology still remain. Moreover, species-specific variation between Plasmodium falciparum and Plasmodium vivax transmission dynamics pose another significant challenge for worldwide malaria elimination efforts. Here, we review the biology of transmission stages, highlighting numerous factors influencing development and dynamics of gametocytes within the host and determinants of human infectiousness.