Due to a more physiological reproduction of the in vivo situation, liver‐on‐a‐chip devices in recent studies have shown higher sensitivity and accuracy for hepatotoxicity testing compared to traditional in vitro liver models. In this context, this work presents an original microfluidic device mimicking 3D hepatic cords organized radially, like in the liver lobule. Ten cell‐culture chambers seeded at a high density are disposed with a parallelized flow that emulates blood flow from the portal triad to the central vein in the liver lobule. Using this device, on‐chip differentiation of human HepaRG‐Hepatoblast (HepaRG‐HB) to HepaRG‐Hepatocytes (HepaRG‐HC) has been studied in terms of self‐organization capabilities, bile canaliculi formation and albumin expression. Our results indicate that due to the design of the cell culture chamber, which mechanically constrains tissue proliferation, and the physiologically relevant microfluidic flow condition used during tissue development, the HepaRG‐HBs in the device can proliferate, self‐organize, and spontaneously differentiate in a DMSO free medium forming long, directional bile canaliculi. On the contrary, under the same conditions, differentiated HepaRG‐HC is observed to aggregate without long bile canaliculi and form tissues resembling traditional HepaRG‐HC cultures. © 2024 Institute of Electrical Engineers of Japan. Published by Wiley Periodicals LLC.