The identification of stem cell-specific proteins and the elucidation of their novel regulatory pathways may help in the development of protocols for control of their self-renewal and differentiation for cell-based therapies. Nucleostemin is a recently discovered nucleolar protein predominantly associated with proliferating rat neural and embryonic stem cells, and some human cancer cell lines. A comprehensive study of nucleostemin in human adult bone marrow stem cells is lacking. The aim of the study was to determine if nucleostemin is synthesized by adult bone marrow stem cells and to analyze its expression during their expansion and differentiation. Using a multipotential adherent population of stem cells, nucleostemin was localized to the nucleoli and occurred in 43.3% of the cells. There was a high level of expression of nucleostemin mRNA in bone marrow stem cells and this remained unchanged over time during cell expansion in culture. When bone marrow stem cells were stimulated to proliferate by fibroblast growth factor (FGF)-2, nucleostemin expression increased in a dose-dependent manner. Small interfering RNA (siRNA) knockdown of nucleostemin abolished the proliferative effect of FGF-2. When bone marrow stem cells were differentiated into chondrocytes, adipocytes, or osteocytes, nucleostemin expression was 70%-90% lower than in the undifferentiated cells retained in monolayer culture. We conclude that nucleostemin is a marker of undifferentiated human adult bone marrow stem cells and that it is involved in the regulation of proliferation of these cells.