Background/Objectives: We aimed to identify the molecular signatures of primitive CD34⁺ and CD34⁻ hematopoietic stem/progenitor cell (HSC/HPC) subsets in cord blood and bone marrow samples. Methods: CD34⁺ and CD34⁻ HSC/HPC subsets from cord blood and bone marrow were characterized using flow cytometry, real-time PCR, and proteomic analysis to evaluate their phenotypic and molecular profiles. Results: Our findings revealed a significantly higher percentage of Lin⁻CD34⁻CD38^Low/− (−/−) cells than of Lin⁻CD34⁺CD38^Low/− (+/−) cells in cord blood. Aldehyde dehydrogenase levels were significantly lower in (−/−) than in (+/−) cells. Clonogenic ability was lower in (−/−) than in (+/−) cells. However, CD34⁻ cells exhibited potent megakaryocyte/erythrocyte differentiation ability. Importantly, the HSC/HPC subsets expressed pluripotency or stemness genes (SOX2, Nanog, and OCT4); however, OCT4 expression significantly increased in (−/−) compared with (+/−) cells. We identified 304 proteins in the HSC/HPC subsets—85.6% had similar expression patterns in the two subsets; only 14.4% were differentially expressed between (−/−) and (+/−) cells. This implies their comparability at the protein level. Certain proteins were implicated in cellular-development-, gene-expression-, and embryonic-development-related signaling networks. Conclusions: Distinct biological and functional characteristics were observed between (−/−) and (+/−) HSC/HPC subsets. Some of the identified proteins may be novel HSC/HPC subsets markers for clinical applications after validation.