Tsetse flies (Diptera: Glossinidae) are important agricultural and medical vectors transmitting the African trypanosomes, the agents of sleeping sickness disease in humans and various diseases in animals (nagana). While the prevalence of disease has increased to epidemic proportions, lack of a mammalian vaccine and affordable and effective drugs have hindered disease control. Trypanosomiasis management relies heavily on the control of its single insect vector, the tsetse fly. Despite the effectiveness of some of these tools, their impact on disease control has not been sustainable due to their local nature and extensive dependence on community participation. Recent advances in molecular technologies and their application to insects have revolutionized the field of vector biology, and there is hope that such new approaches may form the basis for future tsetse interventions. The success of the genetic approaches aiming to disrupt the transmission cycle of the parasite in their invertebrate host depends on full understanding of the interaction between tsetse and trypanosomes. This article reviews the biology of trypanosome development in the fly and the multiple bacterial symbionts that inhabit the same gut environment. The availability of a genetic transformation system for the midgut symbiont allows for gene products to be expressed in vivo in the tsetse gut where they can produce a hostile environment for pathogen transmission. The characterization of gene product(s) with anti-pathogenic properties and their expression in vivo is discussed. A strategy is outlined where the replacement of susceptible insect phenotypes with their engineered refractory counterparts can result in decreased disease transmission.