Probiotics are live, nonpathogenic microorganisms known to have a positive effect on the host by improving the natural balance of gut microbiota. The objective of this study was to determine the effects of administering probiotics (Primalac W/S) in ovo on hatchability, early post-hatch performance, and intestinal immune-related gene expression of broiler chicks. At embryonic day eighteen, 360 Cobb 500 eggs were injected with sterile water (sham), 1 × 105, 1 × 106, or 1 × 107 (P1, P2, and P3 respectively) probiotic bacteria. Another 90 eggs remained non-injected to serve as a negative control. Measurements and tissue samples were taken on day of hatch (DOH) and days 4, 6, 8, 15, and 22. No significant differences were seen among groups for hatchability, feed intake, feed conversion ratios, or mortality. Body weight of P2 was significantly greater than that of the negative control, sham and P1 on d 4, and that of the negative control and P1 on d 6. A similar pattern was observed for BW gain (BWG) from DOH to d 4. Real-time PCR was used to investigate the expression of immune-related genes in the ileum and cecal tonsils. Other than an initial upregulation of inducible nitric oxide synthase on DOH, in ovo probiotic supplementation was associated with downregulated expression of Toll-like receptors-2 and -4, inducible nitric oxide synthase, trefoil factor-2, mucin-2, interferon-γ, and interleukins-4 and -13 in both the ileum and cecal tonsils, though expression patterns differed based on treatment, tissue, and time point evaluated. Taken together, these results indicate that in ovo supplementation of the probiotic product Primalac does not impact hatchability, can improve performance during the first week post-hatch, and is capable of modulating gene expression in the ileum and cecal tonsils.