Recent reports demonstrated that mesenchymal stem cells (MSCs) can induce differentiation of neuroblastoma (NB) cells. Dedifferentiated fat cells (DFAT) and MSCs have similar properties. The present study investigated whether DFAT can induce NB cell differentiation and suppress cell proliferation. DFAT was obtained from mature adipocytes isolated from adipose tissue from a ceiling culture. NB cells were cultured in a medium with/without DFAT, and subsequently in a DFAT-conditioned medium (CM) with/without phosphatidylinositol 3 kinase (PI3K) inhibitor. Length of neurites was measured, and the mRNA expression levels of the neurofilament (NF) and tubulin beta III (TUBβ3) were assessed using quantitative real-time reverse transcription polymerase chain reaction. Cell viability was assessed by the water-soluble tetrazolium salt-1 assay. NB cells cultured with DFAT elongated the neurites and upregulated the expression of NF and Tubβ3 compared with the control. However, NB cells cultured in DFAT-CM demonstrated increased cell viability compared with the control. NB cells cultured with DFAT-CM and PI3K inhibitor suppressed cell viability and demonstrated increased neurite length and expression and upregulation of Tubβ3. Therefore, the combined use of DFAT-CM and PI3K inhibitors suppresses the proliferation of NB cells and induces their differentiation. DFAT may offer new insights into therapeutic approaches in NB.