Human embryonic stem (hES) cells have the potential to serve as an unlimited source of cells for transplantation therapies. However, controlling their proliferation and differentiation into complex, viable three-dimensional (3D) tissues remains a challenge. This study explores the use of biodegradable polymer scaffolds to promote hES cell growth, differentiation, and the formation of 3D structures. The research demonstrates that complex structures with features of various committed embryonic tissues can be generated in vitro using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment, such as poly(lactic-co-glycolic acid)/poly(l-lactic acid) (PLGA/PLLA) polymer scaffolds. Growth factors such as retinoic acid, transforming growth factor β, activin-A, and insulin-like growth factor were found to influence hES cell differentiation, leading to the formation of 3D structures with characteristics of developing neural tissues, cartilage, liver, and blood vessels. When transplanted into severe combined immunodeficient (SCID) mice, the constructs continued to express specific human proteins in defined differentiated structures and appeared to recruit and anastomose with the host vasculature. The study highlights the potential of polymer scaffolds as a unique culture system for addressing questions in cell and developmental biology and for creating viable human tissue structures for therapeutic applications. The results indicate that complex structures with features of various committed embryonic tissues can be generated in vitro using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment such as PLGA/PLLA polymer scaffolds. The in vivo results show that scaffold-supported hES constructs remain viable for at least 2 weeks, that constructs may recruit and anastomose with the host vascular system, and that the differentiation pattern induced in vitro remains intact or continues to progress in vivo. The study also emphasizes the importance of further research to promote tissue differentiation in vitro and in vivo and to address the regulation of cellular proliferation to allay concerns regarding the potential tumorigenicity of undifferentiated and precursor cells.Human embryonic stem (hES) cells have the potential to serve as an unlimited source of cells for transplantation therapies. However, controlling their proliferation and differentiation into complex, viable three-dimensional (3D) tissues remains a challenge. This study explores the use of biodegradable polymer scaffolds to promote hES cell growth, differentiation, and the formation of 3D structures. The research demonstrates that complex structures with features of various committed embryonic tissues can be generated in vitro using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment, such as poly(lactic-co-glycolic acid)/poly(l-lactic acid) (PLGA/PLLA) polymer scaffolds. Growth factors such as retinoic acid, transforming growth factor β, activin-A, and insulin-like growth factor were found to influence hES cell differentiation, leading to the formation of 3D structures with characteristics of developing neural tissues, cartilage, liver, and blood vessels. When transplanted into severe combined immunodeficient (SCID) mice, the constructs continued to express specific human proteins in defined differentiated structures and appeared to recruit and anastomose with the host vasculature. The study highlights the potential of polymer scaffolds as a unique culture system for addressing questions in cell and developmental biology and for creating viable human tissue structures for therapeutic applications. The results indicate that complex structures with features of various committed embryonic tissues can be generated in vitro using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment such as PLGA/PLLA polymer scaffolds. The in vivo results show that scaffold-supported hES constructs remain viable for at least 2 weeks, that constructs may recruit and anastomose with the host vascular system, and that the differentiation pattern induced in vitro remains intact or continues to progress in vivo. The study also emphasizes the importance of further research to promote tissue differentiation in vitro and in vivo and to address the regulation of cellular proliferation to allay concerns regarding the potential tumorigenicity of undifferentiated and precursor cells.