Neural stem cell line developed from human umbilical cord blood (HUCB-NSC) [Buzańska et al., 2003. Journal of Neurochemistry 85, 33] is an ethically uncontroversial source of stem cells, able to differentiate into neuronal, astrocytic and oligodendroglial lineages. Developmental fate decisions of HUCB-NSC can be experimentally manipulated in vitro by the presence of trophic factors, mitogenes and neuromorphogenes, but can also be influenced by neurotoxins. In this report two-dimensional (2-D) and three-dimensional (3-D) HUCB-NSC cultures are introduced as useful models for testing developmental neurotoxicity. For 2-D culture models we established a standardized method for the assessment of the growth rate and cell differentiation in 96-well plates. The proliferative capacity of the HUCB-NSC was monitored by the MTT test while their ability to differentiate into neural-like cells by immunocytochemistry of beta-tubulin III and MAP-2 for neurons, GFAP and S-100beta for astrocytes and GalC for oligodendrocytes. The 3-D culture of HUCB-NSC is represented by neurospheres. Proliferation and migration of the intermediate precursors from attached neurospheres are shown to be controlled and altered by various growth factors and further modulated by the extracellular matrix component-fibronectin. Thus, neurospheres derived from the HUCB-NSC line can represent a suitable model of the activation of dormant stem cells residing in their niche, and can be used for neurotoxic studies.