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Simple Summary: We previously demonstrated that cells of cows produced from reconstructed embryos containing the cytoplasm of a heat-tolerant breed (Bos indicus, BI) showed improved thermotolerance despite their nuclei being derived from a heat-sensitive breed (Bos Taurus, BT). In this study, we utilized spindle transfer (ST) technology to transfer karyoplasts from heat-sensitive Holstein cattle (H) into recipient ooplasm from heat-tolerant Taiwan yellow cattle (Y). This process resulted in the creation of ST oocytes (ST-Yo-Hn). These ST oocytes were subsequently subjected to in vitro fertilization (IVF) with Holstein bull semen, resulting in fertilized ST embryos. The resulting ST-Yo-Hn blastocysts were then transferred into recipient females, leading to the successful production of three ST-Yo-Hn cattle. To understand the factors mediating thermotolerance, ear fibroblasts from ST-Yo-Hn and H cattle were isolated, and their differentially expressed protein and gene profiles under heat shock (42 °C for 12 h) were compared. The results indicated that the ear fibroblasts of the ST-Yo-Hn cattle exhibited significantly (p < 0.05) lower expression of pro-apoptotic factors, higher expression of anti-apoptotic factors, and higher antioxidant ability under heat stress, making the somatic cells from the ST-Yo-Hn cattle more heat-tolerant than those from the H cattle. Global warming has a significant impact on the dairy farming industry, as heat stress causes reproductive endocrine imbalances and leads to substantial economic losses, particularly in tropical–subtropical regions. The Holstein breed, which is widely used for dairy production, is highly susceptible to heat stress, resulting in a dramatic reduction in milk production during hot seasons. However, previous studies have shown that cells of cows produced from reconstructed embryos containing cytoplasm (o) from Taiwan yellow cattle (Y) have improved thermotolerance despite their nuclei (n) being derived from heat-sensitive Holstein cattle (H). Using spindle transfer (ST) technology, we successfully produced ST-Yo-Hn cattle and proved that the thermotolerance of their ear fibroblasts is similar to that of Y and significantly better than that of H (p < 0.05). Despite these findings, the genes and molecules responsible for the different sensitivities of cells derived from ST-Yo-Hn and H cattle have not been extensively investigated. In the present study, ear fibroblasts from ST-Yo-Hn and H cattle were isolated, and differentially expressed protein and gene profiles were compared with or without heat stress (hs) (42 °C for 12 h). The results revealed that the relative protein expression levels of pro-apoptotic factors, including Caspase-3, -8, and -9, in the ear fibroblasts from the ST-Yo-Hn-hs group were significantly lower (p < 0.05) than those from the H-hs group. Conversely, the relative expression levels of anti-apoptotic factors, including GNA14 protein and the CRELD2 and PRKCQ genes, were significantly higher (p < 0.05) in the ear fibroblasts from the ST-Yo-Hn-hs group compared to those from the H-hs group. Analysis of oxidative phosphorylation-related factors revealed that the relative expression levels of the GPX1 gene and Complex-I, Complex-IV, CAT, and PGLS proteins were significantly higher (p < 0.05) in the ear fibroblasts from the ST-Yo-Hn-hs group compared to those from the H-hs group. Taken together, these findings suggest that ear fibroblasts from ST-Yo-Hn cattle have superior thermotolerance compared to those from H cattle due to their lower expression of pro-apoptotic factors and higher expression of oxidative phosphorylation and antioxidant factors. Moreover, this improved thermotolerance is attributed, at least partially, to the cytoplasm derived from more heat-tolerant Y cattle. Hence, using ST technology to produce more heat-tolerant H cattle containing Y cytoplasm could be a feasible approach to alleviate the negative impacts of heat stress on dairy cattle in tropical–subtropical regions. [ABSTRACT FROM AUTHOR]