This study introduces a novel ex vivo model for colorectal oncogenesis, called mini-colons, which are three-dimensional organoid cultures that can undergo tumorigenesis. The mini-colons are engineered using microfabrication, optogenetics, and tissue engineering techniques, allowing for spatiotemporal control of oncogenic activation through blue-light exposure. This system enables the real-time tracking of tumor development at the single-cell level for several weeks without the need for passaging. The induced mini-colons exhibit intratumoral and intertumoral diversity, recapitulating key pathophysiological hallmarks of colorectal tumors in vivo. By fine-tuning cell-intrinsic and cell-extrinsic parameters, the mini-colons can be used to identify tumor-initiating factors and pharmacological targets. The study demonstrates that the mini-colon system provides a high-resolution, in vitro platform for studying cancer initiation, bridging the gap between in vitro and in vivo models. The findings highlight the potential of mini-colons as a versatile tool for screening cellular and molecular determinants of cancer development, reducing the reliance on animal models in cancer research.This study introduces a novel ex vivo model for colorectal oncogenesis, called mini-colons, which are three-dimensional organoid cultures that can undergo tumorigenesis. The mini-colons are engineered using microfabrication, optogenetics, and tissue engineering techniques, allowing for spatiotemporal control of oncogenic activation through blue-light exposure. This system enables the real-time tracking of tumor development at the single-cell level for several weeks without the need for passaging. The induced mini-colons exhibit intratumoral and intertumoral diversity, recapitulating key pathophysiological hallmarks of colorectal tumors in vivo. By fine-tuning cell-intrinsic and cell-extrinsic parameters, the mini-colons can be used to identify tumor-initiating factors and pharmacological targets. The study demonstrates that the mini-colon system provides a high-resolution, in vitro platform for studying cancer initiation, bridging the gap between in vitro and in vivo models. The findings highlight the potential of mini-colons as a versatile tool for screening cellular and molecular determinants of cancer development, reducing the reliance on animal models in cancer research.