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Galactooligosaccharides (GOS) that are delivered in ovo improve intestinal microbiota composition and mitigate the negative effects of heat stress in broiler chickens. Hubbard hybrids are slow-growing chickens with a high resistance to heat. In this paper, we determined the impact of GOS delivered in ovo on slow-growing chickens that are challenged with heat. The experiment was a 2 &#215; 2 &#215; 2 factorial design. On day 12 of incubation, GOS (3.5 mg/egg) was delivered into the egg (n = 300). Controls (C) were mock-injected with physiological saline (n = 300). After hatching, the GOS and C groups were split into thermal groups: thermoneutral (TN) and heat stress (HS). HS (30 &#176;C) lasted for 14 days (days 36&#8722;50 post-hatching). The spleen (n = 8) was sampled after acute (8.5 h) and chronic (14 days) HS. The gene expression of immune-related (<i>IL-2, IL-4, IL-6, IL-10, IL-12p40,</i> and <i>IL-17</i>) and stress-related genes (<i>HSP25, HSP90AA1, BAG3, CAT,</i> and <i>SOD</i>) was detected with RT-qPCR. Chronic HS up-regulated the expression of the genes: <i>IL-10</i>, <i>IL-12p40</i>, <i>SOD</i> (<i>p</i> &lt; 0.05), and <i>CAT</i> (<i>p</i> &lt; 0.01). GOS delivered in ovo down-regulated <i>IL-4</i> (acute <i>p</i> &lt; 0.001; chronic <i>p</i> &lt; 0.01), <i>IL-12p40</i>, <i>CAT</i> and <i>SOD</i> (chronic <i>p</i> &lt; 0.05). The obtained results suggest that slow-growing hybrids are resistant to acute heat and tolerant to chronic heat, which can be supported with in ovo GOS administration.