Neurotrophic factors (NF) are critical for neuronal survival, differentiation, and regeneration. We investigated the effects of Noggin, FGF-9, and GDNF on neurite outgrowth and proliferation using a three-dimensional (3D) model of neural spheroids (NS) derived from corpus striatum (CS) stem cells of E14 rat embryos. NS were cultured in suspension and embedded in type I collagen to assess NF effects at 24–48 h. Proliferation was analyzed with the MTT assay, while immunocytochemistry was performed for Ki67, Nestin, TubIII, c-FOS, and GFAP. Clear NS formation was observed, confirming stem cell properties. NF stimulation significantly enhanced neurite extension compared to controls, with Noggin showing the strongest effect on axonal elongation. After 48 h, stimulated cultures displayed higher expression of neuronal and glial markers, indicating differentiation. Conversely, FGF-9 predominantly promoted NS proliferation in suspensión cultures. These findings demonstrate that the 3D collagen assay supports NSC proliferation and differentiation with high reproducibility, providing a reliable platform to study NF action in regenerative medicine.