Mycoplasma gallisepticum (MG) is a bacterium that causes egg production losses in layer chickens raised on multi-age layer hen complexes. To combat this loss, layer chickens are vaccinated against MG before they are moved to the layer facility to begin laying eggs. The objective of this dissertation research was to investigate the potential of the in ovo vaccination of layer chickens against field strain MG infections to enhance immunity, and reduce labor and material costs associated with the current post-hatch vaccination method. Initial studies tested various dosages of a live attenuated strain F (FMG) vaccine delivered in ovo by hand injection at 18 days of incubation. The greatest dilution of vaccine (1-3 colony forming units (CFU) per dose) did not have an adverse effect on hatch success and was able to induce initial antibody production against FMG in approximately 50% of the birds raised through 6 weeks of age, with relatively low posthatch mortality. Higher dosages tested, starting at 102 CFU per dose, caused extremely high (>50%) post-hatch mortality during the first 2 weeks and were considered impractical. These in ovo-vaccinated birds, even at the lowest FMG dose, were able to transmit the bacteria to other MG-clean birds with which they were in direct contact. The lowest in ovo FMG dose was further tested for its ability to be applied using a current in ovo vaccination machine. This machine externally disinfects each injection needle after every injection. The FMG was detected in FMG-vaccinated birds at 6 weeks of age whether or not they had received the disinfection step. Furthermore, birds hatched from eggs that were injected with and without disinfection had comparable humoral immune responses against FMG, with similar results to the hand injection study. Thus, the disinfection step during in ovo vaccination caused no loss of FMG vaccine efficiency and the in ovo vaccination of layer chickens against FMG could be readily practiced in the poultry industry. Future work should evaluate how this in ovo vaccination regimen compares with other post-hatch MG vaccination regimens for layer chickens through the lay cycle and against a field strain MG challenge.