Commercial layer hens reared on multi-age hen complexes are vaccinated during pullet rearing to combat production losses due to the bacteria Mycoplasma gallisepticum (MG). In this study, the potential to in ovo vaccinate layer chickens against MG was investigated. Layer embryos were administered a dosage of a live attenuated strain F MG (FMG) vaccine at 18 d of incubation and raised for 6 wk for initial post-hatch evaluation in 2 replicate trials. Treatments included control non-injected eggs, eggs injected with diluent, a non-diluted dosage, a 10-2 dilution, a 10-4 dilution, and a 10-6 dilution. A subset of chicks were swabbed for detection of FMG in the trachea at hatch. At 6 wk of age, birds were swabbed again for FMG detection and a blood sample was tested for MG antibody production. Hatch was depressed in the non-diluted dose group (P < 0.0001). Strain F MG was detected at hatch in the trachea in each FMG injection treatment, with decreasing numbers of positive chicks in the lower dosage groups. Mortality during the first 2 wk post-hatch was 3.5% (trial 1) and was 11.7% (trial 2) in the 10-6 dilution treatment, with all other FMG treatments experiencing a high rate of mortality (>50%). Birds in the in ovo FMG treatments had detectable FMG and antibody production at 6 wk. There were no differences in percentage positive birds (P > 0.3 for all tests) or ELISA titers (P = 0.079) between the FMG treatments. Body weight at 6 wk of age was diminished with increasing FMG dose (P < 0.0001). The lowest dose tested was found to be the most practical, causing the least mortality, least weight loss, and a humoral immune response in the majority of the birds. Further work is needed to evaluate how this in ovo vaccine, promoting immunity earlier, would compare to a standard post-hatch vaccination against an MG challenge scenario through a lay cycle.