During the first week post-hatch, the neonatal chick is immunologically vulnerable and subject to infectious threats found in the environment. Probiotics are live, non-pathogenic microorganisms known to have a positive effect on the host by improving the natural balance of gut microbiota and promoting animal health. The objective of this study was to determine the effects of administering probiotics in ovo and in the diet on broiler chick hatchability, and post-hatch immune organ weights and ileal immune-related gene expression. At embryonic day 18, 1584 eggs were injected with nothing (Dry), 1 × 10 6 , or 1 × 10 7 (P1 and P2 respectively) probiotic bacteria. The remaining 393 eggs were left non-injected to serve as a negative control. Immune organ weights and tissue samples were taken on DOH and d4, 6, 8, 14, and 20. No differences were observed for hatchability or relative bursa weights. Only on d6, the P2 birds receiving the probiotic-supplemented diet had larger spleens as a result of a 2-way interaction between in ovo treatment and post-hatch diet. Both in ovo and dietary administration of probiotics were able to modulate the expression of the immune-related genes in the ileum; however, expression patterns differed based on the gene, treatment, and time point evaluated. In conclusion, these results indicate that in ovo supplementation of this commercial probiotic product does not influence hatchability and is capable of differentially modulating expression of certain genes in the ileum. Furthermore, in ovo administration of probiotics has an effect similar to that of dietary supplementation endorsing its usage to potentially promote early colonization of beneficial bacteria to stimulate intestinal and immune system development.