In mouse tooth development, the roots of the first lower molar develop after crown formation to form 2 cylindrical roots by post-natal day 5. This study compared the morphogenesis and cellular events of the mesial-root-forming (MRF) and bifurcation-forming (BF) regions, located in the mesial and center of the first lower molar, to better define the developmental mechanisms involved in multi-rooted tooth formation. We found that the mesenchyme in the MRF showed relatively higher proliferation than the bifurcation region. This suggested that spatially regulated mesenchymal proliferation is required for creating cylindrical root structure. The mechanism may involve the mesenchyme forming a physical barrier to epithelial invagination of Hertwig’s epithelial root sheath. To test these ideas, we cultured roots in the presence of pharmacological inhibitors of microtubule and actin polymerization, nocodazole and cytochalasin-D. Cytochalasin D also inhibits proliferation in epithelium and mesenchyme. Both drugs resulted in altered morphological changes in the tooth root structures. In particular, the nocodazole- and cytochalasin-D-treated specimens showed a loss of root diameter and formation of a single-root, respectively. Immunolocalization and three-dimensional reconstruction results confirmed these mesenchymal cellular events, with higher proliferation in MRF in multi-rooted tooth formation.