Colorectal cancer (CRC) remains the 3rd cause of cancer-related death suggesting that currently available treatments are unable to eliminate all cancer cells. Tumor growth and behavior is determined by a small population of cancer stem cells able to proliferate extensively. These cells are also able to differentiate into the heterogeneous groups of cells forming the tumor, are resistant to current therapies and thus responsible for recurrence. Recent reports suggest that the stem cell populations located at basis of the intestinal crypt are susceptible to become cancer-initiating cells. The homeostasis of these crypt stem cells is tightly controlled by their environment, which includes lumenal content, as well as cellular microenvironment (fibroblasts, macrophages, etc.). Here, using several models including the human 3D colon organoid model, we investigated the effects of microenvironmental proteases, and investigated the differential role of the protease-activated receptors 1 and 2 (PAR1 and PAR2) on the crypt cells regulation. We also investigated the role of PAR1 and PAR2 in the activation of normal colon fibroblasts, an event known to be of bad prognostic for CRC patients. Taken together, data will be presented on the contribution of proteolytic balance to stem cell biology and implications for colorectal cancer.