Cells invade extracellular matrices in a regulated manner at specific times and places during normal development. A dramatic example is trophoblast invasion of the uterine wall. Previous studies have shown that differentiation of trophoblasts to an invasive phenotype is accompanied by temporally and spatially regulated switching of their integrin repertoire. In the first trimester human placenta, alpha 6 integrins are restricted to cytotrophoblast (CTB) stem cells and downregulated in invasive CTBs, whereas alpha 5 beta 1 and alpha 1 beta 1 integrins are upregulated in differentiating and invasive CTBs. The goal of the present study was to determine whether these changes have functional consequences for CTB invasiveness. Using an in vitro invasion model, we determined first that aggregates of invading first trimester CTBs in vitro undergo the same pattern of integrin switching as was observed in situ, thereby validating the utility of the model. We then showed that antibody perturbation of interactions involving laminin or collagen type IV and their integrin alpha 1/beta 1 receptor inhibited invasion by CTBs, whereas perturbing interactions between fibronectin and the alpha 5/beta 1 fibronectin receptor accelerated invasion. Finally, we report that later gestation CTBs, which display greatly decreased invasive capacity, are also unable to upregulate alpha 1 beta 1 complexes, providing further evidence that this integrin is critical for CTB invasion. This gestational regulation is transcriptional. These data indicate that integrin switching observed during differentiation in situ has significant functional consequences for CTB invasion. The data suggest further that differentiating CTBs upregulate counterbalancing invasion-accelerating and invasion-restraining adhesion mechanisms. We propose that this contributes to regulating the depth of CTB invasion during normal implantation.