Background: Mesenchymal stem cells (MSCs) exhibit a high capacity for differentiation, possess anti-inflammatory and proangiogenic properties, and stimulate the growth and proliferation of neighboring cells. MSCs are a promising tool in regenerative medicine. However, the molecular mechanisms underlying the properties of these cells are not yet fully understood. Gene expression in MSCs influences their characteristics and differentiation potential. Therefore, it is essential to investigate factors affecting gene expression as well as those activating signaling pathways, which will enable more effective and individualized applications of MSCs. In this study, we aimed to identify signaling pathways involved in gene expression in umbilical cord-derived MSCs (UC-MSCs) that may be altered by maternal diabetes and hypothyroidism during pregnancy. Methods: The research material consisted of UC-MSCs. Samples obtained from nine participants were analyzed. UC-MSCs were isolated and cultured, and RNA was extracted. The isolated RNA was used for microarray-based gene expression analysis. Subsequently, pathway enrichment analysis was performed to identify the signaling pathways involved. Results: In the diabetes group, 340 genes (0.71%) were upregulated, while 268 genes (0.56%) were downregulated compared with UC-MSCs from the control group. In the diabetes group, the most compact module was composed of proteins associated with WNT/planar cell polarity (WNT/PCP) signaling. The second module included genes related to smooth muscle activity. In the hypothyroidism group, an association was identified between the extracellular matrix organization pathways (GO:0030198) and the extracellular structure organization (GO:0043062) pathways. Moreover, in this group, increased expression of MMP1, MMP10, and GREM1 was observed. Conclusions: In summary, our study demonstrated the impact of diabetes and hypothyroidism on gene expression in UC-MSCs. We also observed the activation of distinct signaling pathways depending on the presence of these conditions. However, this work represents a preliminary screening, and the results should be validated by PCR in a larger cohort.