Abstract Objective: This study aimed to evaluate neuronal markers in stromal cells from human exfoliated deciduous teeth (SHED) and standardize the isolation and characterization of those cells. Methodology: Healthy primary teeth were collected from children. The cells were isolated by enzymatic digestion with collagenase. By following the International Society for Cell and Gene Therapy (ISCT) guidelines, SHED were characterized by flow cytometry and differentiated into osteogenic, adipogenic, and chondrogenic lineages. Colony-forming unit-fibroblasts (CFU-F) were performed to assess these cells’ potential and efficiency. To clarify the neuronal potential of SHED, the expression of nestin and βIII-tubulin were examined by immunofluorescence and SOX1, SOX2, GFAP, and doublecortin (DCX), nestin, CD56, and CD146 by flow cytometry. Results: SHED showed mesenchymal stromal cells characteristics, such as adhesion to plastic, positive immunophenotypic profile for CD29, CD44, CD73, CD90, CD105, and CD166 markers, reduced expression for CD14, CD19, CD34, CD45, HLA-DR, and differentiation in three lineages confirmed by staining and gene expression for adipogenic differentiation. The average efficiency of colony formation was 16.69%. SHED expressed the neuronal markers nestin and βIII-tubulin; the fluorescent signal intensity was significantly higher in βIII-tubulin (p<0.0001) compared to nestin. Moreover, SHED expressed DCX, GFAP, nestin, SOX1, SOX2, CD56, CD146, and CD271. Therefore, SHED had a potential for neuronal lineage even without induction with culture medium and specific factors. Conclusion: SHEDs may be a new therapeutic strategy for regenerating and repairing neuronal cells and tissues. Objective (SHED Methodology children collagenase ISCT (ISCT guidelines osteogenic Colonyforming Colony forming unitfibroblasts unit fibroblasts CFUF CFU F (CFU-F βIIItubulin βIII tubulin SOX1 SOX SOX2 GFAP DCX , (DCX) CD56 CD CD14 Results characteristics plastic CD29 CD44 CD73 CD90 CD105 CD16 CD19 CD34 CD45 HLADR, HLADR HLA DR, DR HLA-DR 1669 16 69 16.69% p<0.0001 p00001 p 0 0001 (p<0.0001 Moreover CD271 Therefore factors Conclusion tissues (DCX CD5 CD1 CD2 CD4 CD7 CD9 CD10 CD3 166 1 6 16.69 p<0.000 p0000 000 (p<0.000 CD27 16.6 p<0.00 p000 00 (p<0.00 16. p<0.0 p00 (p<0.0 p<0. p0 (p<0. p<0 (p<0 p< (p< (p