Photosynthetic microalgae continue to be of great interest as a promising class of feedstock for biofuels and higher-value chemicals and oils, with the potential for contributing to liquid transportation fuel supplies while reducing GHG emissions through the displacement of fossil petroleum-based fuel and chemical product usage. However, to significantly contribute to fuel supplies will require algae cultivation scale-up to large aggregated quantities of biomass and fuel feedstock production that will necessarily impose huge demands for land, water, supplemental CO2, and other key macronutrients such as nitrogen and phosphorus. The resulting resource requirements will impose constraints on the level of production that can be sustainably supported. This presentation provides a high-level analysis and review of the current status and future prospects for these key resource demand and constraint challenges for the scale-up of autotrophic microalgal biofuel production. The analysis is focused on resource demand issues and assumes that the current techno-economic challenges facing the affordable scale-up of algal biofuels production can be overcome. Emphasis is placed on the USA, although the issues are generally relevant globally. Developing microalgae production approaches that can effectively use non-fresh water resources, capture and recycle both water and macronutrients from downstream algae harvesting and processing, and minimize requirements for both fresh make-up water and commercial nutrients will help reduce, but not eliminate, resource constraints. Providing supplemental CO2 resources for enhanced and affordable algae production may be the biggest challenge. Based on current understanding, resource demand could constrain U.S. algal biofuel production to levels below 10 BGY.