The indefinite proliferative capacity and ability to differentiate into all somatic cell types can make human embryonic stem cells (hESCs) useful in experimental and applied studies in embryonic development, tissue engineering, genetic engineering, pharmacokinetics, and the like. Cellular differentiation dynamics can be studied in monolayer cell cultures; however, it proceeds in three-dimensional (3D) organization in vivo. The aim of this study was to assess the effects of retinoic acid ( RA) and nerve growth factor (NGF) on the differentiation patterns of hESCs in 3D culture environment and to compare it with the monolayer culture. Expanded hESCs (HUES-9) were differentiated in two experimental groups for 21 days: (i) two-dimensional (2D) monolayer cultures of hESC colonies, and (ii) 3D culture of hES single cells in poly(DL-lactic-co-glycolic acid) scaffolds. The media used were embryonic stem cell expansion medium (ES-EM), embryonic stem cell differentiation medium containing fetal calf serum (ES-DM), ES-EM containing either 10 ng/mL NGF or 10(-6) M RA, and their combination. Fixed specimens were analyzed with scanning electron microscopy, and expression of nestin, pan-cytokeratin, troponin, and alpha-fetoprotein at days 7, 14, and 21 was evaluated by immunohistomorphometry and reverse transcriptase polymerase chain reaction. Results indicate different patterns of ectodermal, mesodermal, and endodermal marker expressions between groups, where NGF and RA preferentially favors the differentiation toward ectodermal and mesodermal lineages. While troponin and nestin expression is significantly elevated in 3D culture environment, pan-cytokeratin expression is favored by 2D culture instead. The effects of 3D scaffold culture imply the usefulness of testing in vitro differentiation properties of hESCs in various culture settings designed as models in prospective tissue engineering applications.