A new type of mesenchymal stem cells (MSCs) isolated from mastoid bone marrow obtained during mastoidectomies were identified. The isolated cells were analyzed by fluorescence-activated cell sorting (FACS) using CD45, CD90, CD105, and CD29 for investigation of the characteristics of the isolated cells. Stem cell phenotypes of human mastoid bone marrow-derived cells (hMBMCs) were maintained during passages 0–4. In the proliferation profiles of stem cells, FACS showed positive results for CD90, CD105, CD29, and negative for CD45. We found that the cells differentiated into adipocytes, chondrocytes, osteocytes, neuronal, and epithelial cells. The morphological features of the differentiated cells were also observed. Our results demonstrated that hMBMCs showed general features of MSCs that can differentiate into multiple cell types under specific stimulation, which could be used as a valuable source of MSCs for various applications in tissue engineering and regenerative medicine, especially for otolaryngology such as one stage reconstruction of functional ears.